Introduction of HIV-2 and multiple HIV-1 subtypes to Lebanon.

HIV genetic variability, phylogenetic relationships, and transmission dynamics were analyzed in 26 HIV-infected patients from Lebanon. Twenty-five specimens were identified as HIV-1 and one as HIV-2 subtype B. The 25 strains were classified into six env-C2-V3 HIV-1 subtypes: B (n = 10), A (n = 11), C (n = 1), D (n = 1), G (n = 1), and unclassifiable. Potential recombinants combining parts of viral regions from different subtypes Aenv/Dpol/Agag, Genv/Apol, and the unclassifiable-subtype(env)/unclassifiable-subtype(pol)/Agag were found in three patients. Epidemiologic analysis of travel histories and behavioral risks indicated that HIV-1 and HIV-2 subtypes reflected HIV strains prevalent in countries visited by patients or their sex partners. Spread of complex HIV-subtype distribution patterns to regions where HIV is not endemic may be more common than previously thought. Blood screening for both HIV-1 and HIV-2 in Lebanon is recommended to protect the blood supply. HIV subtype data provide information for vaccine development

Measuring Enzymatic HIV-1 Susceptibility to Two Reverse Transcriptase Inhibitors as a Rapid and Simple Approach to HIV-1 Drug-Resistance Testing

Simple and cost-effective approaches for HIV drug-resistance testing are highly desirable for managing increasingly expanding HIV-1 infected populations who initiate antiretroviral therapy (ART), particularly in resource-limited settings. Non-nucleoside reverse trancriptase inhibitor (NNRTI)-based regimens with an NRTI backbone containing lamivudine (3TC) or emtricitabine (FTC) are preferred first ART regimens. Failure with these drug combinations typically involves the selection of NNRTI- and/or 3TC/FTC- resistant viruses. Therefore, the availability of simple assays to measure both types of drug resistance is critical. We have developed a high throughput screening test for assessing enzymatic resistance of the HIV-1 RT in plasma to 3TC/FTC and NNRTIs. The test uses the sensitive “Amp-RT” assay with a newly-developed real-time PCR format to screen biochemically for drug resistance in single reactions containing either 3TC-triphosphate (3TC-TP) or nevirapine (NVP). Assay cut-offs were defined based on testing a large panel of subtype B and non-subtype B clinical samples with known genotypic profiles. Enzymatic 3TC resistance correlated well with the presence of M184I/V, and reduced NVP susceptibility was strongly associated with the presence of K103N, Y181C/I, Y188L, and G190A/Q. The sensitivity and specificity for detecting resistance were 97.0% and 96.0% in samples with M184V, and 97.4% and 96.2% for samples with NNRTI mutations, respectively. We further demonstrate the utility of an HIV capture method in plasma by using magnetic beads coated with CD44 antibody that eliminates the need for ultracentifugation. Thus our results support the use of this simple approach for distinguishing WT from NNRTI- or 3TC/FTC-resistant viruses in clinical samples. This enzymatic testing is subtype-independent and can assist in the clinical management of diverse populations particularly in resource-limited settings

1970s and 'Patient 0' HIV-1 genomes illuminate early HIV/AIDS history in North America.

The emergence of HIV-1 group M subtype B in North American men who have sex with men was a key turning point in the HIV/AIDS pandemic. Phylogenetic studies have suggested cryptic subtype B circulation in the United States (US) throughout the 1970s and an even older presence in the Caribbean. However, these temporal and geographical inferences, based upon partial HIV-1 genomes that postdate the recognition of AIDS in 1981, remain contentious and the earliest movements of the virus within the US are unknown. We serologically screened >2,000 1970s serum samples and developed a highly sensitive approach for recovering viral RNA from degraded archival samples. Here, we report eight coding-complete genomes from US serum samples from 1978-1979-eight of the nine oldest HIV-1 group M genomes to date. This early, full-genome 'snapshot' reveals that the US HIV-1 epidemic exhibited extensive genetic diversity in the 1970s but also provides strong evidence for its emergence from a pre-existing Caribbean epidemic. Bayesian phylogenetic analyses estimate the jump to the US at around 1970 and place the ancestral US virus in New York City with 0.99 posterior probability support, strongly suggesting this was the crucial hub of early US HIV/AIDS diversification. Logistic growth coalescent models reveal epidemic doubling times of 0.86 and 1.12 years for the US and Caribbean, respectively, suggesting rapid early expansion in each location. Comparisons with more recent data reveal many of these insights to be unattainable without archival, full-genome sequences. We also recovered the HIV-1 genome from the individual known as 'Patient 0' (ref. 5) and found neither biological nor historical evidence that he was the primary case in the US or for subtype B as a whole. We discuss the genesis and persistence of this belief in the light of these evolutionary insights.This work was supported by NIH/NIAID R01AI084691 and the David and Lucile Packard Foundation (M.W.); the Wellcome Trust (080651), the University of Oxford’s Clarendon Fund, the Economic and Social Research Council (PTA-026-27-2838), and a J. Armand Bombardier Internationalist Fellowship (R.A.M.); the Research Fund KU Leuven (Onderzoeksfonds KU Leuven, Program Financing no. PF/10/018) and the ‘Fonds voor Wetenschappelijk Onderzoek Vlaanderen’ (FWO) (G066215N) (P.L); and NSF DMS 1264153, NIH R01 HG006139 and NIH R01 AI107034 (M.A.S.).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/nature1982

Absence of evidence of Xenotropic Murine Leukemia Virus-related virus infection in persons with Chronic Fatigue Syndrome and healthy controls in the United States

<p>Abstract</p> <p>Background</p> <p>XMRV, a xenotropic murine leukemia virus (MuLV)-related virus, was recently identified by PCR testing in 67% of persons with chronic fatigue syndrome (CFS) and in 3.7% of healthy persons from the United States. To investigate the association of XMRV with CFS we tested blood specimens from 51 persons with CFS and 56 healthy persons from the US for evidence of XMRV infection by using serologic and molecular assays. Blinded PCR and serologic testing were performed at the US Centers for Disease Control and Prevention (CDC) and at two additional laboratories.</p> <p>Results</p> <p>Archived blood specimens were tested from persons with CFS defined by the 1994 international research case definition and matched healthy controls from Wichita, Kansas and metropolitan, urban, and rural Georgia populations. Serologic testing at CDC utilized a Western blot (WB) assay that showed excellent sensitivity to MuLV and XMRV polyclonal or monoclonal antibodies, and no reactivity on sera from 121 US blood donors or 26 HTLV-and HIV-infected sera. Plasma from 51 CFS cases and plasma from 53 controls were all WB negative. Additional blinded screening of the 51 cases and 53 controls at the Robert Koch Institute using an ELISA employing recombinant Gag and Env XMRV proteins identified weak seroreactivity in one CFS case and a healthy control, which was not confirmed by immunofluorescence. PCR testing at CDC employed a <it>gag </it>and a <it>pol </it>nested PCR assay with a detection threshold of 10 copies in 1 ug of human DNA. DNA specimens from 50 CFS patients and 56 controls and 41 US blood donors were all PCR-negative. Blinded testing by a second nested gag PCR assay at the Blood Systems Research Institute was also negative for DNA specimens from the 50 CFS cases and 56 controls.</p> <p>Conclusions</p> <p>We did not find any evidence of infection with XMRV in our U.S. study population of CFS patients or healthy controls by using multiple molecular and serologic assays. These data do not support an association of XMRV with CFS.</p

Simple PCR Assays Improve the Sensitivity of HIV-1 Subtype B Drug Resistance Testing and Allow Linking of Resistance Mutations

The success of antiretroviral therapy is known to be compromised by drug-resistant HIV-1 at frequencies detectable by conventional bulk sequencing. Currently, there is a need to assess the clinical consequences of low-frequency drug resistant variants occurring below the detection limit of conventional genotyping. Sensitive detection of drug-resistant subpopulations, however, requires simple and practical methods for routine testing.We developed highly-sensitive and simple real-time PCR assays for nine key drug resistance mutations and show that these tests overcome substantial sequence heterogeneity in HIV-1 clinical specimens. We specifically used early wildtype virus samples from the pre-antiretroviral drug era to measure background reactivity and were able to define highly-specific screening cut-offs that are up to 67-fold more sensitive than conventional genotyping. We also demonstrate that sequencing the mutation-specific PCR products provided a direct and novel strategy to further detect and link associated resistance mutations, allowing easy identification of multi-drug-resistant variants. Resistance mutation associations revealed in mutation-specific amplicon sequences were verified by clonal sequencing.Combined, sensitive real-time PCR testing and mutation-specific amplicon sequencing provides a powerful and simple approach that allows for improved detection and evaluation of HIV-1 drug resistance mutations

Failure to Detect the Novel Retrovirus XMRV in Chronic Fatigue Syndrome

BACKGROUND:In October 2009 it was reported that 68 of 101 patients with chronic fatigue syndrome (CFS) in the US were infected with a novel gamma retrovirus, xenotropic murine leukaemia virus-related virus (XMRV), a virus previously linked to prostate cancer. This finding, if confirmed, would have a profound effect on the understanding and treatment of an incapacitating disease affecting millions worldwide. We have investigated CFS sufferers in the UK to determine if they are carriers of XMRV. METHODOLOGY:Patients in our CFS cohort had undergone medical screening to exclude detectable organic illness and met the CDC criteria for CFS. DNA extracted from blood samples of 186 CFS patients were screened for XMRV provirus and for the closely related murine leukaemia virus by nested PCR using specific oligonucleotide primers. To control for the integrity of the DNA, the cellular beta-globin gene was amplified. Negative controls (water) and a positive control (XMRV infectious molecular clone DNA) were included. While the beta-globin gene was amplified in all 186 samples, neither XMRV nor MLV sequences were detected. CONCLUSION:XMRV or MLV sequences were not amplified from DNA originating from CFS patients in the UK. Although we found no evidence that XMRV is associated with CFS in the UK, this may be a result of population differences between North America and Europe regarding the general prevalence of XMRV infection, and might also explain the fact that two US groups found XMRV in prostate cancer tissue, while two European studies did not

Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs

The xenotropic murine leukemia virus (MLV)-related viruses (XMRV) have been reported in persons with prostate cancer, chronic fatigue syndrome, and less frequently in blood donors. Polytropic MLVs have also been described in persons with CFS and blood donors. However, many studies have failed to confirm these findings, raising the possibility of contamination as a source of the positive results. One PCR reagent, Platinum Taq polymerase (pol) has been reported to contain mouse DNA that produces false-positive MLV PCR results. We report here the finding of a large number of PCR reagents that have low levels of MLV sequences. We found that recombinant reverse-transcriptase (RT) enzymes from six companies derived from either MLV or avian myeloblastosis virus contained MLV pol DNA sequences but not gag or mouse DNA sequences. Sequence and phylogenetic analysis showed high relatedness to Moloney MLV, suggesting residual contamination with an RT-containing plasmid. In addition, we identified contamination with mouse DNA and a variety of MLV sequences in commercially available human DNAs from leukocytes, brain tissues, and cell lines. These results identify new sources of MLV contamination and highlight the importance of careful pre-screening of commercial specimens and diagnostic reagents to avoid false-positive MLV PCR results