Mutagenesis of Murine Cytomegalovirus Using a Tn3-Based Transposon

AbstractA transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals

High Frequency of Syncytium-Inducing and CXCR4-Tropic Viruses among Human Immunodeficiency Virus Type 1 Subtype C-Infected Patients Receiving Antiretroviral Treatment

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism

Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes

CITATION: Duong, Y. T., et al. 2015. Recalibration of the limiting antigen avidity EIA to determine mean duration of recent infection in divergent HIV-1 subtypes. PLoS ONE, 10(2):1-15, doi:10.1371/journal.pone.0114947.The original publication is available at http://journals.plos.org/plosoneBackground: Mean duration of recent infection (MDRI) and misclassification of long-term HIV-1 infections, as proportion false recent (PFR), are critical parameters for laboratory-based assays for estimating HIV-1 incidence. Recent review of the data by us and others indicated that MDRI of LAg-Avidity EIA estimated previously required recalibration. We present here results of recalibration efforts using >250 seroconversion panels and multiple statistical methods to ensure accuracy and consensus. Methods: A total of 2737 longitudinal specimens collected from 259 seroconverting individuals infected with diverse HIV-1 subtypes were tested with the LAg-Avidity EIA as previously described. Data were analyzed for determination of MDRI at ODn cutoffs of 1.0 to 2.0 using 7 statistical approaches and sub-analyzed by HIV-1 subtypes. In addition, 3740 specimens from individuals with infection >1 year, including 488 from patients with AIDS, were tested for PFR at varying cutoffs. Results: Using different statistical methods, MDRI values ranged from 88–94 days at cutoff ODn = 1.0 to 177–183 days at ODn = 2.0. The MDRI values were similar by different methods suggesting coherence of different approaches. Testing for misclassification among long-term infections indicated that overall PFRs were 0.6% to 2.5% at increasing cutoffs of 1.0 to 2.0, respectively. Balancing the need for a longer MDRI and smaller PFR (<2.0%) suggests that a cutoff ODn = 1.5, corresponding to an MDRI of 130 days should be used for cross-sectional application. The MDRI varied among subtypes from 109 days (subtype A&D) to 152 days (subtype C). Conclusions: Based on the new data and revised analysis, we recommend an ODn cutoff = 1.5 to classify recent and long-term infections, corresponding to an MDRI of 130 days (118–142). Determination of revised parameters for estimation of HIV-1 incidence should facilitate application of the LAg-Avidity EIA for worldwide usehttp://journals.plos.org/plosone/article?id=10.1371/journal.pone.0114947Publisher's versio

Implementation of a Test, Treat, and Prevent HIV program among men who have sex with men and transgender women in Thailand, 2015-2016

<div><p>Introduction</p><p>Antiretroviral therapy reduces the risk of serious illness among people living with HIV and can prevent HIV transmission. We implemented a Test, Treat, and Prevent HIV Program among men who have sex with men (MSM) and transgender women at five hospitals in four provinces of Thailand to increase HIV testing, help those who test positive start antiretroviral therapy, and increase access to pre-exposure prophylaxis (PrEP).</p><p>Methods</p><p>We implemented rapid HIV testing and trained staff on immediate antiretroviral initiation at the five hospitals and offered PrEP at two hospitals. We recruited MSM and transgender women who walked-in to clinics and used a peer-driven intervention to expand recruitment. We used logistic regression to determine factors associated with prevalent HIV infection and the decision to start antiretroviral therapy and PrEP.</p><p>Results</p><p>During 2015 and 2016, 1880 people enrolled. Participants recruited by peers were younger (p<0.0001), less likely to be HIV-infected (p<0.0001), and those infected had higher CD4 counts (p = 0.04) than participants who walked-in to the clinics. Overall, 16% were HIV-positive: 18% of MSM and 9% of transgender women; 86% started antiretroviral therapy and 46% of eligible participants started PrEP. A higher proportion of participants at hospitals with one-stop HIV services started antiretroviral therapy than other hospitals. Participants who started PrEP were more likely to report sex with an HIV-infected partner (p = 0.002), receptive anal intercourse (p = 0.02), and receiving PrEP information from a hospital (p<0.0001).</p><p>Conclusions</p><p>We implemented a Test, Treat, and Prevent HIV Program offering rapid HIV testing and immediate access to antiretroviral therapy and PrEP. Peer-driven recruitment reached people at high risk of HIV and people early in HIV illness, providing an opportunity to promote HIV prevention services including PrEP and early antiretroviral therapy. Sites with one-stop HIV services had a higher uptake of antiretroviral therapy and PrEP.</p></div

Results of logistic regression analysis evaluating characteristics of HIV-uninfected participants in the test, treat, and prevent HIV program at Lerdsin and Thammasat University Hospitals who chose to start HIV pre-exposure prophylaxis (PrEP), Thailand, 2015–2016.

<p>Results of logistic regression analysis evaluating characteristics of HIV-uninfected participants in the test, treat, and prevent HIV program at Lerdsin and Thammasat University Hospitals who chose to start HIV pre-exposure prophylaxis (PrEP), Thailand, 2015–2016.</p

Results of logistic regression analysis to evaluate characteristics of participants in the test, treat, and prevent HIV program associated with prevalent HIV infection, Thailand, 2015–2016.

<p>Results of logistic regression analysis to evaluate characteristics of participants in the test, treat, and prevent HIV program associated with prevalent HIV infection, Thailand, 2015–2016.</p

Knowledge and attitudes of test, treat, and prevent HIV program participants about HIV infection, antiretrovirals, and pre-exposure prophylaxis, Thailand, 2015–2016.

<p>Knowledge and attitudes of test, treat, and prevent HIV program participants about HIV infection, antiretrovirals, and pre-exposure prophylaxis, Thailand, 2015–2016.</p

Results of logistic regression analysis evaluating characteristics of participants in the test, treat, and prevent HIV program with prevalent HIV infection who chose to start antiretroviral therapy (ART), Thailand, 2015–2016.

<p>Results of logistic regression analysis evaluating characteristics of participants in the test, treat, and prevent HIV program with prevalent HIV infection who chose to start antiretroviral therapy (ART), Thailand, 2015–2016.</p

Baseline characteristics of participants in the test, treat, and prevent HIV program, Thailand, 2015–2016.

<p>Baseline characteristics of participants in the test, treat, and prevent HIV program, Thailand, 2015–2016.</p