Publications by authors named "Shuntai Zhou"

30 Publications

  • Page 1 of 1

β-D-N 4-hydroxycytidine (NHC) Inhibits SARS-CoV-2 Through Lethal Mutagenesis But Is Also Mutagenic To Mammalian Cells.

J Infect Dis 2021 May 7. Epub 2021 May 7.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA.

Mutagenic ribonucleosides can act as broad-based antiviral agents. They are metabolized to the active ribonucleoside triphosphate form and concentrate in the genomes of RNA viruses during viral replication. β-D-N 4-hydroxycytidine (NHC, the initial metabolite of molnupiravir) is more than 100-fold more active than ribavirin or favipiravir against SARS-CoV-2, with antiviral activity correlated to the level of mutagenesis in virion RNA. However, NHC also displays host mutational activity in an animal cell culture assay, consistent with RNA and DNA precursors sharing a common intermediate of a ribonucleoside diphosphate. These results indicate that highly active mutagenic ribonucleosides may hold risk for the host.
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http://dx.doi.org/10.1093/infdis/jiab247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8136050PMC
May 2021

Primer ID Next-Generation Sequencing for the Analysis of a Broad Spectrum Antiviral Induced Transition Mutations and Errors Rates in a Coronavirus Genome.

Bio Protoc 2021 Mar 5;11(5):e3938. Epub 2021 Mar 5.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, USA.

Next generations sequencing (NGS) has become an important tool in biomedical research. The Primer ID approach combined with the MiSeq platform overcomes the limitation of PCR errors and reveals the true sampling depth of population sequencing, making it an ideal tool to study mutagenic effects of potential broad-spectrum antivirals on RNA viruses. In this report we describe a protocol using Primer ID sequencing to study the mutations induced by antivirals in a coronavirus genome from an cell culture model and an mouse model. Viral RNA or total lung tissue RNA is tagged with Primer ID-containing cDNA primers during the initial reverse transcription step, followed by two rounds of PCR to amplify viral sequences and incorporate sequencing adaptors. Purified and pooled libraries are sequenced using the MiSeq platform. Sequencing data are processed using the template consensus sequence (TCS) web-app. The Primer ID approach provides an accurate sequencing protocol to measure mutation error rates in viral RNA genomes and host mRNA. Sequencing results suggested that β-D-N4-hydroxycytidine (NHC) greatly increased the transition substitution rate but not the transversion substitution rate in the viral RNA genomes, and cytosine (C) to uridine (U) was found as the most frequently seen mutation.
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http://dx.doi.org/10.21769/BioProtoc.3938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8005877PMC
March 2021

The HIV-1 latent reservoir is largely sensitive to circulating T cells.

Elife 2020 10 6;9. Epub 2020 Oct 6.

Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, United States.

HIV-1-specific CD8+ T cells are an important component of HIV-1 curative strategies. Viral variants in the HIV-1 reservoir may limit the capacity of T cells to detect and clear virus-infected cells. We investigated the patterns of T cell escape variants in the replication-competent reservoir of 25 persons living with HIV-1 (PLWH) durably suppressed on antiretroviral therapy (ART). We identified all reactive T cell epitopes in the HIV-1 proteome for each participant and sequenced HIV-1 outgrowth viruses from resting CD4+ T cells. All non-synonymous mutations in reactive T cell epitopes were tested for their effect on the size of the T cell response, with a≥50% loss defined as an escape mutation. The majority (68%) of T cell epitopes harbored no detectable escape mutations. These findings suggest that circulating T cells in PLWH on ART could contribute to control of rebound and could be targeted for boosting in curative strategies.
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http://dx.doi.org/10.7554/eLife.57246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7593086PMC
October 2020

Two Genetic Differences between Closely Related Zika Virus Strains Determine Pathogenic Outcome in Mice.

J Virol 2020 09 29;94(20). Epub 2020 Sep 29.

Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Recent Zika virus (ZIKV) outbreaks and unexpected clinical manifestations of ZIKV infection have prompted an increase in ZIKV-related research. Here, we identify two strain-specific determinants of ZIKV virulence in mice. We found that strain H/PF/2013 caused 100% lethality in mice, whereas PRVABC59 caused no lethality; both strains caused 100% lethality in double-knockout (DKO) mice. Deep sequencing revealed a high-frequency variant in PRVABC59 not present in H/PF/2013: a G-to-T change at nucleotide 1965 producing a Val-to-Leu substitution at position 330 of the viral envelope (E) protein. We show that the V330 variant is lethal on both virus strain backgrounds, whereas the L330 variant is attenuating only on the PRVABC59 background. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. The consensus sequences of H/PF/2013 and PRVABC59 differ by 3 amino acids, but these were not responsible for the difference in virulence between the two strains. H/PF/2013 and PRVABC59 differ by an additional 31 noncoding or silent nucleotide changes. We made a panel of chimeric viruses with identical amino acid sequences but nucleotide sequences derived from H/PF/2013 or PRVABC59. We found that 6 nucleotide differences in the 3' quarter of the H/PF/2013 genome were sufficient to confer virulence in mice. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis ( and DKO mice). Contemporary ZIKV strains are closely related and often used interchangeably in laboratory research. Here, we identify two strain-specific determinants of ZIKV virulence that are evident in only mice but not DKO mice. These results identify a balanced polymorphism in the E protein that is sufficient to attenuate the PRVABC59 strain but not H/PF/2013. We further identify a second virulence determinant in the H/PF/2013 strain, which is driven by the viral nucleotide sequence but not the amino acid sequence. Altogether, our work identifies a large and previously unreported difference in virulence between two commonly used ZIKV strains, in two widely used mouse models of ZIKV pathogenesis. Our results highlight that even very closely related virus strains can produce significantly different pathogenic phenotypes in common laboratory models.
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http://dx.doi.org/10.1128/JVI.00618-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527068PMC
September 2020

Fact and Fiction about 1%: Next Generation Sequencing and the Detection of Minor Drug Resistant Variants in HIV-1 Populations with and without Unique Molecular Identifiers.

Viruses 2020 08 4;12(8). Epub 2020 Aug 4.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Next generation sequencing (NGS) platforms have the ability to generate almost limitless numbers of sequence reads starting with a PCR product. This gives the illusion that it is possible to analyze minor variants in a viral population. However, including a PCR step obscures the sampling depth of the viral population, the key parameter needed to understand the utility of the data set for finding minor variants. Also, these high throughput sequencing platforms are error prone at the level where minor variants are of interest, confounding the interpretation of detected minor variants. A simple strategy has been applied in multiple applications of NGS to solve these problems. Prior to PCR, individual molecules are "tagged" with a unique molecular identifier (UMI) that can be used to establish the actual sample size of viral genomes sequenced after PCR and sequencing. In addition, since PCR generates many copies of each sequence tagged to a specific UMI, a template consensus sequence (TCS) can be created from the many reads of each template, removing virtually all of the method error. From this perspective we examine our own use of a UMI, called Primer ID, in the detection of minor drug resistant variants in HIV-1 populations.
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http://dx.doi.org/10.3390/v12080850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472098PMC
August 2020

Near Real-Time Identification of Recent Human Immunodeficiency Virus Transmissions, Transmitted Drug Resistance Mutations, and Transmission Networks by Multiplexed Primer ID-Next-Generation Sequencing in North Carolina.

J Infect Dis 2021 Mar;223(5):876-884

University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

Background: The identification of recent human immunodeficiency virus (HIV) 1 infections among people with new HIV diagnoses is important to both tailoring and assessing the impact of HIV-1 prevention strategies.

Methods: We developed a multiplexed Primer ID-next-generation sequencing approach to identify recent infections by measuring the intrahost viral diversity over multiple regions of the HIV-1 genome, in addition to detecting drug resistance mutations (DRMs) and phylogenetically linked clusters. We summarize the field implementation of this all-in-one platform among persons with newly diagnosed HIV-1 by the North Carolina State Laboratory of Public Health in 2018.

Results: Overall, recent infection was identified in 94 (35%) of 268 patients with new HIV diagnoses. People <30 years old, and people who inject drugs were more likely to have diagnoses of recent infection. The reverse-transcriptase region K103N was the most commonly detected DRM (prevalence, approximately 15%). We found a total of 28 clusters, and persons with recent infection were more likely to be cluster members than were those with chronic infections (P = .03).

Conclusions: We demonstrate the rapid identification of recent infection and pretreatment DRMs coupled with cluster analysis that will allow prioritization of linkage to care, treatment, and prevention interventions to those at highest risk of onward transmission.
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http://dx.doi.org/10.1093/infdis/jiaa417DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7938176PMC
March 2021

Multi-Laboratory Comparison of Next-Generation to Sanger-Based Sequencing for HIV-1 Drug Resistance Genotyping.

Viruses 2020 06 27;12(7). Epub 2020 Jun 27.

Rush Medical College, Chicago, IL 60612, USA.

Next-generation sequencing (NGS) is increasingly used for HIV-1 drug resistance genotyping. NGS methods have the potential for a more sensitive detection of low-abundance variants (LAV) compared to standard Sanger sequencing (SS) methods. A standardized threshold for reporting LAV that generates data comparable to those derived from SS is needed to allow for the comparability of data from laboratories using NGS and SS. Ten HIV-1 specimens were tested in ten laboratories using Illumina MiSeq-based methods. The consensus sequences for each specimen using LAV thresholds of 5%, 10%, 15%, and 20% were compared to each other and to the consensus of the SS sequences (protease 4-99; reverse transcriptase 38-247). The concordance among laboratories' sequences at different thresholds was evaluated by pairwise sequence comparisons. NGS sequences generated using the 20% threshold were the most similar to the SS consensus (average 99.6% identity, range 96.1-100%), compared to 15% (99.4%, 88.5-100%), 10% (99.2%, 87.4-100%), or 5% (98.5%, 86.4-100%). The average sequence identity between laboratories using thresholds of 20%, 15%, 10%, and 5% was 99.1%, 98.7%, 98.3%, and 97.3%, respectively. Using the 20% threshold, we observed an excellent agreement between NGS and SS, but significant differences at lower thresholds. Understanding how variation in NGS methods influences sequence quality is essential for NGS-based HIV-1 drug resistance genotyping.
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http://dx.doi.org/10.3390/v12070694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411816PMC
June 2020

Deep Sequencing Reveals Compartmentalized HIV-1 in the Semen of Men with and without Sexually Transmitted Infection-Associated Urethritis.

J Virol 2020 06 1;94(12). Epub 2020 Jun 1.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Concurrent sexually transmitted infections (STI) can increase the probability of HIV-1 transmission primarily by increasing the viral load present in semen. In this study, we explored the relationship of HIV-1 in blood and seminal plasma in the presence and absence of urethritis and after treatment of the concurrent STI. Primer ID deep sequencing of the V1/V3 region of the HIV-1 gene was done for paired blood and semen samples from antiretroviral therapy (ART)-naive men living in Malawi with ( = 19) and without ( = 5) STI-associated urethritis; for a subset of samples, full-length genes were generated for sequence analysis and to test entry phenotype. Cytokine concentrations in the blood and semen were also measured, and a reduction in the levels of proinflammatory cytokines was observed following STI treatment. We observed no difference in the prevalence of diverse compartmentalized semen-derived lineages in men with or without STI-associated urethritis, and these viral populations were largely stable during STI treatment. Clonal amplification of one or a few viral sequences accounted for nearly 50% of the viral population, indicating a recent bottleneck followed by limited viral replication. We conclude that the male genital tract is a site where virus can be brought in from the blood, where localized sustained replication can occur, and where specific genotypes can be amplified, perhaps initially by cellular proliferation but further by limited viral replication. HIV-1 infection is a sexually transmitted infection that coexists with other STI. Here, we examined the impact of a concurrent STI resulting in urethritis on the HIV-1 population within the male genital tract. We found that viral populations remain largely stable even with treatment of the STI. These results show that viral populations within the male genital tract are defined by factors beyond transient inflammation associated with a concurrent STI.
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http://dx.doi.org/10.1128/JVI.00151-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307086PMC
June 2020

An orally bioavailable broad-spectrum antiviral inhibits SARS-CoV-2 in human airway epithelial cell cultures and multiple coronaviruses in mice.

Sci Transl Med 2020 04 6;12(541). Epub 2020 Apr 6.

Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Coronaviruses (CoVs) traffic frequently between species resulting in novel disease outbreaks, most recently exemplified by the newly emerged SARS-CoV-2, the causative agent of COVID-19. Here, we show that the ribonucleoside analog β-d-N-hydroxycytidine (NHC; EIDD-1931) has broad-spectrum antiviral activity against SARS-CoV-2, MERS-CoV, SARS-CoV, and related zoonotic group 2b or 2c bat-CoVs, as well as increased potency against a CoV bearing resistance mutations to the nucleoside analog inhibitor remdesivir. In mice infected with SARS-CoV or MERS-CoV, both prophylactic and therapeutic administration of EIDD-2801, an orally bioavailable NHC prodrug (β-d-N-hydroxycytidine-5'-isopropyl ester), improved pulmonary function and reduced virus titer and body weight loss. Decreased MERS-CoV yields in vitro and in vivo were associated with increased transition mutation frequency in viral, but not host cell RNA, supporting a mechanism of lethal mutagenesis in CoV. The potency of NHC/EIDD-2801 against multiple CoVs and oral bioavailability highlights its potential utility as an effective antiviral against SARS-CoV-2 and other future zoonotic CoVs.
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http://dx.doi.org/10.1126/scitranslmed.abb5883DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7164393PMC
April 2020

HIV-1 Central Nervous System Compartmentalization and Cytokine Interplay in Non-Subtype B HIV-1 Infections in Nigeria and Malawi.

AIDS Res Hum Retroviruses 2020 06 17;36(6):490-500. Epub 2020 Feb 17.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

HIV-1 compartmentalization in the central nervous system (CNS) and its contribution to neurological disease have been well documented. Previous studies were conducted among people infected with subtypes B or C where CNS compartmentalization has been observed when comparing viral sequences in the blood to virus in cerebrospinal fluid (CSF). However, little is known about CNS compartmentalization in other HIV-1 subtypes. Using a deep sequencing approach with Primer ID, we conducted a cross-sectional study among Nigerian and Malawian HIV-1 cohorts with or without fungal Cryptococcus infection diagnosed as cryptococcal meningitis (CM) to determine the extent of CSF/CNS compartmentalization with CM. Paired plasma and CSF samples from 45 participants were also analyzed for cytokine/chemokine levels. Viral populations comparing virus in the blood and the CSF ranged from compartmentalized to equilibrated, including minor or partial compartmentalization or clonal amplification of a single viral sequence. The frequency of compartmentalized viral populations in the blood and CSF was similar between the CM- and CM+ participants. We confirmed the potential to see compartmentalization with subtype C infection and have also documented CNS compartmentalization of an HIV-1 subtype G infection. Cytokine profiles indicated a proinflammatory environment, especially within the CSF/CNS. However, sCD163 was suppressed in the CSF in the presence of CM, perhaps due to elevated levels of IL-4, which were also a feature of the cytokine profile, showing a distinct cytokine profile with CM.
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http://dx.doi.org/10.1089/AID.2019.0245DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262640PMC
June 2020

The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation.

Sci Transl Med 2019 10;11(513)

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4 T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4 T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.
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http://dx.doi.org/10.1126/scitranslmed.aaw5589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7233356PMC
October 2019

Picomolar to Micromolar: Elucidating the Role of Distal Mutations in HIV-1 Protease in Conferring Drug Resistance.

ACS Chem Biol 2019 11 13;14(11):2441-2452. Epub 2019 Aug 13.

Department of Biochemistry and Molecular Pharmacology , University of Massachusetts Medical School , Worcester , Massachusetts 01605 , United States.

Drug resistance continues to be a growing global problem. The efficacy of small molecule inhibitors is threatened by pools of genetic diversity in all systems, including antibacterials, antifungals, cancer therapeutics, and antivirals. Resistant variants often include combinations of active site mutations and distal "secondary" mutations, which are thought to compensate for losses in enzymatic activity. HIV-1 protease is the ideal model system to investigate these combinations and underlying molecular mechanisms of resistance. Darunavir (DRV) binds wild-type (WT) HIV-1 protease with a potency of <5 pM, but we have identified a protease variant that loses potency to DRV 150 000-fold, with 11 mutations in and outside the active site. To elucidate the roles of these mutations in DRV resistance, we used a multidisciplinary approach, combining enzymatic assays, crystallography, and molecular dynamics simulations. Analysis of protease variants with 1, 2, 4, 8, 9, 10, and 11 mutations showed that the primary active site mutations caused ∼50-fold loss in potency (2 mutations), while distal mutations outside the active site further decreased DRV potency from 13 nM (8 mutations) to 0.76 μM (11 mutations). Crystal structures and simulations revealed that distal mutations induce subtle changes that are dynamically propagated through the protease. Our results reveal that changes remote from the active site directly and dramatically impact the potency of the inhibitor. Moreover, we find interdependent effects of mutations in conferring high levels of resistance. These mechanisms of resistance are likely applicable to many other quickly evolving drug targets, and the insights may have implications for the design of more robust inhibitors.
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http://dx.doi.org/10.1021/acschembio.9b00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6941144PMC
November 2019

Evolutionary pathways to NS5A inhibitor resistance in genotype 1 hepatitis C virus.

Antiviral Res 2018 10 3;158:45-51. Epub 2018 Aug 3.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Division of Infectious Diseases, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA. Electronic address:

Direct-acting antivirals (DAAs) targeting NS5A are broadly effective against hepatitis C virus (HCV) infections, but sustained virological response rates are generally lower in patients infected with genotype (gt)-1a than gt-1b viruses. The explanation for this remains uncertain. Here, we adopted a highly accurate, ultra-deep primer ID sequencing approach to intensively study serial changes in the NS5A-coding region of HCV in gt-1a- and gt-1b-infected subjects receiving a short course of monotherapy with the NS5A inhibitor, elbasvir. Low or undetectable levels of viremia precluded on-treatment analysis in gt-1b-infected subjects, but variants with the resistance-associated substitution (RAS) Y93H in NS5A dominated rebounding virus populations following cessation of treatment. These variants persisted until the end of the study, two months later. In contrast, while Y93H emerged in multiple lineages and became dominant in subjects with gt-1a virus, these haplotypes rapidly decreased in frequency off therapy. Substitutions at Q30 and L31 emerged in distinctly independent lineages at later time points, ultimately coming to dominate the virus population off therapy. Consistent with this, cell culture studies with gt-1a and gt-1b reporter viruses and replicons demonstrated that Y93H confers a much greater loss of replicative fitness in gt-1a than gt-1b virus, and that L31M/V both compensates for the loss of fitness associated with Q30R (but not Y93H) and also boosts drug resistance. These observations show how differences in the impact of RASs on drug resistance and replicative fitness influence the evolution of gt-1a and gt-1b viruses during monotherapy with an antiviral targeting NS5A.
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http://dx.doi.org/10.1016/j.antiviral.2018.07.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6146077PMC
October 2018

Using Primer-ID Deep Sequencing to Detect Recent Human Immunodeficiency Virus Type 1 Infection.

J Infect Dis 2018 10;218(11):1777-1782

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill.

Intrahost viral sequence diversity can be evaluated over multiple genomic regions using next-generation sequencing (NGS) and scaled to population-level diversity to identify recent human immunodeficiency virus type 1 infection. Using Primer-ID NGS, we sequenced the reverse transcriptase (RT) and env V1-V3 regions from persons with known infection dates, and assessed the mean (π) and first quintile of pairwise diversity distributions over time. The receiver operating characteristic area under the curve (AUC) of RT and V1-V3 combined showed excellent discrimination of recent infection <9 months: using π (only single transmitted variants: AUC, 0.98; threshold <1.03%; sensitivity, 97%; specificity, 89%) and the first quintile (including all variants: AUC, 0.90; threshold <0.60%; sensitivity, 91%; specificity, 92%).
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http://dx.doi.org/10.1093/infdis/jiy426DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6195657PMC
October 2018

Characterization of the Transmitted Virus in an Ongoing HIV-1 Epidemic Driven by Injecting Drug Use.

AIDS Res Hum Retroviruses 2018 10 30;34(10):867-878. Epub 2018 Jul 30.

1 Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill , Chapel Hill, North Carolina.

Understanding features of the HIV-1 transmission process has the potential to inform biological interventions for prevention. We have examined the transmitted virus in a cohort of people who inject drugs and who are at risk of HIV-1 infection through blood contamination when injecting in a group. This study focused on seven newly infected participants in St. Petersburg, Russia, who were in acute or early infection. We used end-point dilution polymerase chain reaction to amplify single viral genomes to assess the complexity of the transmitted virus. We also used deep sequencing to further assess the complexity of the virus. We interpret the results as indicating that a single viral variant was transmitted in each case, consistent with a model where the exposure to virus during transmission was limited. We also looked at phenotypic properties of the viral Env protein in isolates from acute and chronic infection. Although differences were noted, there was no consistent pattern that distinguished the transmitted variants. Similarly, despite the reduced genetic heterogeneity of the more recent subtype A HIV-1 epidemic in St. Petersburg, we did not see reduced variance in the neutralization properties compared to isolates from the more mature subtype C HIV-1 epidemic. Finally, in looking at members of injecting groups related to the acute HIV-1 infection/early subjects, we found examples of sequence linkage consistent with ongoing and rapid spread of HIV-1 in these groups. These studies emphasize the dynamic nature of this epidemic and reinforce the idea that improved prevention methods are needed.
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http://dx.doi.org/10.1089/AID.2017.0313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6204568PMC
October 2018

Prevalence of Drug-Resistant Minority Variants in Untreated HIV-1-Infected Individuals With and Those Without Transmitted Drug Resistance Detected by Sanger Sequencing.

J Infect Dis 2017 08;216(3):387-391

Division of Infectious Diseases and Geographic Medicine.

Minority variant human immunodeficiency virus type 1 (HIV-1) nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance mutations are associated with an increased risk of virological failure during treatment with NNRTI-containing regimens. To determine whether individuals to whom variants with isolated NNRTI-associated drug resistance were transmitted are at increased risk of virological failure during treatment with a non-NNRTI-containing regimen, we identified minority variant resistance mutations in 33 individuals with isolated NNRTI-associated transmitted drug resistance and 49 matched controls. We found similar proportions of overall and nucleoside reverse transcriptase inhibitor-associated minority variant resistance mutations in both groups, suggesting that isolated NNRTI-associated transmitted drug resistance may not be a risk factor for virological failure during treatment with a non-NNRTI-containing regimen.
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http://dx.doi.org/10.1093/infdis/jix338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5853472PMC
August 2017

Sequencing the Biology of Entry: The Retroviral env Gene.

Curr Top Microbiol Immunol 2017;407:65-82

Department of Biochemistry and Biophysics, Lineberger Cancer Center, University of North Carolina, Chapel Hill, NC, 27599, USA.

The surface envelope protein of any virus is major determinant of the host cell that is infected and as a result a major determinant of viral pathogenesis. Retroviruses have a single surface protein named Env. It is a trimer of heterodimers and is responsible for binding to the host cell receptor and mediating fusion between the viral and host membranes. In this review we will discuss the history of the discovery of the avian leukosis virus (ALV) and human immunodeficiency virus type 1 (HIV-1) Env proteins and their receptor specificity, comparing the many differences but having some similarities. Much of the progress in these fields has relied on viral genetics and genetic polymorphisms in the host population. A special feature of HIV-1 is that its persistent infection in its human host, to the point of depleting its favorite target cells, allows the virus to evolve new entry phenotypes to expand its host range into several new cell types. This variety of entry phenotypes has led to confusion in the field leading to the major form of entry phenotype of HIV-1 being overlooked until recently. Thus an important part of this story is the description and naming of the most abundant entry form of the virus: R5 T cell-tropic HIV-1.
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http://dx.doi.org/10.1007/82_2017_35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122457PMC
June 2019

Characterizing HIV-1 Splicing by Using Next-Generation Sequencing.

J Virol 2017 03 28;91(6). Epub 2017 Feb 28.

UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Full-length human immunodeficiency virus type 1 (HIV-1) RNA serves as the genome or as an mRNA, or this RNA undergoes splicing using four donors and 10 acceptors to create over 50 physiologically relevant transcripts in two size classes (1.8 kb and 4 kb). We developed an assay using Primer ID-tagged deep sequencing to quantify HIV-1 splicing. Using the lab strain NL4-3, we found that A5 (/) is the most commonly used acceptor (about 50%) and A3 () the least used (about 3%). Two small exons are made when a splice to acceptor A1 or A2 is followed by activation of donor D2 or D3, and the high-level use of D2 and D3 dramatically reduces the amount of and transcripts. We observed distinct patterns of temperature sensitivity of splicing to acceptors A1 and A2. In addition, disruption of a conserved structure proximal to A1 caused a 10-fold reduction in all transcripts that utilized A1. Analysis of a panel of subtype B transmitted/founder viruses showed that splicing patterns are conserved, but with surprising variability of usage. A subtype C isolate was similar, while a simian immunodeficiency virus (SIV) isolate showed significant differences. We also observed transsplicing from a downstream donor on one transcript to an upstream acceptor on a different transcript, which we detected in 0.3% of 1.8-kb RNA reads. There were several examples of splicing suppression when the intron was retained in the 4-kb size class. These results demonstrate the utility of this assay and identify new examples of HIV-1 splicing regulation. During HIV-1 replication, over 50 conserved spliced RNA variants are generated. The splicing assay described here uses new developments in deep-sequencing technology combined with Primer ID-tagged cDNA primers to efficiently quantify HIV-1 splicing at a depth that allows even low-frequency splice variants to be monitored. We have used this assay to examine several features of HIV-1 splicing and to identify new examples of different mechanisms of regulation of these splicing patterns. This splicing assay can be used to explore in detail how HIV-1 splicing is regulated and, with moderate throughput, could be used to screen for structural elements, small molecules, and host factors that alter these relatively conserved splicing patterns.
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http://dx.doi.org/10.1128/JVI.02515-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331825PMC
March 2017

Quantification of the Latent HIV-1 Reservoir Using Ultra Deep Sequencing and Primer ID in a Viral Outgrowth Assay.

J Acquir Immune Defic Syndr 2017 Feb;74(2):221-228

*Department of Biochemistry and Biophysics, UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC; †Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC; and Departments of ‡Medicine; §Microbiology and Immunology, UNC HIV Cure Center, University of North Carolina at Chapel Hill, Chapel Hill, NC.

Background: In this study, we measured the latent HIV-1 reservoir harboring replication-competent HIV-1 in resting CD4 T cells in participants on highly active antiretroviral therapy, quantitating the frequency of latent infection through the use of a Primer ID-based Ultra Deep Sequencing Assay (UDSA), in comparison to the readout of the quantitative viral outgrowth assay (QVOA).

Methods: Viral RNA derived from culture wells of QVOA that scored as HIV-1 p24 capsid antigen positive were tagged with a specific barcode during cDNA synthesis, and the sequences within the V1-V3 region of the HIV-1 env gene were analyzed for diversity using the Primer ID-based paired-end MiSeq platform. We analyzed samples from a total of 19 participants, 2 initially treated with highly active antiretroviral therapy in acute infection and 17 treated during chronic infection. Phylogenetic trees were generated with all viral lineages detected from culture wells derived from each participant to determine the number of distinct viral lineages growing out in each well, thus capturing another level of information beyond the well being positive for viral antigen. The infectious units per million (IUPM) cell values estimated using a maximum likelihood approach, based on the number of distinct viral lineages detected (VOA-UDSA), were compared with those obtained from QVOA measured using limiting dilution.

Results: IUPM estimates determined by VOA-UDSA ranged from 0.14 to 3.66 and strongly correlated with the IUPM estimates determined by QVOA (r = 0.94; P < 0.0001).

Conclusions: VOA-UDSA may be an alternative readout for that currently used for QVOA.
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http://dx.doi.org/10.1097/QAI.0000000000001187DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5233602PMC
February 2017

Cytosine deamination and the precipitous decline of spontaneous mutation during Earth's history.

Proc Natl Acad Sci U S A 2016 07 5;113(29):8194-9. Epub 2016 Jul 5.

Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599

The hydrolytic deamination of cytosine and 5-methylcytosine residues in DNA appears to contribute significantly to the appearance of spontaneous mutations in microorganisms and in human disease. In the present work, we examined the mechanism of cytosine deamination and the response of the uncatalyzed reaction to changing temperature. The positively charged 1,3-dimethylcytosinium ion was hydrolyzed at a rate similar to the rate of acid-catalyzed hydrolysis of 1-methylcytosine, for which it furnishes a satisfactory kinetic model and a probable mechanism. In agreement with earlier reports, uncatalyzed deamination was found to proceed at very similar rates for cytosine, 1-methylcytosine, cytidine, and cytidine 5'-phosphate, and also for cytosine residues in single-stranded DNA generated from a phagemid, in which we sequenced an insert representing the gene of the HIV-1 protease. Arrhenius plots for the uncatalyzed deamination of cytosine were linear over the temperature range from 90 °C to 200 °C and indicated a heat of activation (ΔH(‡)) of 23.4 ± 0.5 kcal/mol at pH 7. Recent evidence indicates that the surface of the earth has been cool enough to support life for more than 4 billion years and that life has been present for almost as long. If the temperature at Earth's surface is assumed to have followed Newton's law of cooling, declining exponentially from 100 °C to 25 °C during that period, then half of the cytosine-deaminating events per unit biomass would have taken place during the first 0.2 billion years, and <99.4% would have occurred during the first 2 billion years.
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http://dx.doi.org/10.1073/pnas.1607580113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4961170PMC
July 2016

Deep Sequencing of the HIV-1 env Gene Reveals Discrete X4 Lineages and Linkage Disequilibrium between X4 and R5 Viruses in the V1/V2 and V3 Variable Regions.

J Virol 2016 08 27;90(16):7142-58. Epub 2016 Jul 27.

UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA UNC Center for AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Unlabelled: HIV-1 requires the CD4 receptor and a coreceptor (CCR5 [R5 phenotype] or CXCR4 [X4 phenotype]) to enter cells. Coreceptor tropism can be assessed by either phenotypic or genotypic analysis, the latter using bioinformatics algorithms to predict tropism based on the env V3 sequence. We used the Primer ID sequencing strategy with the MiSeq sequencing platform to reveal the structure of viral populations in the V1/V2 and C2/V3 regions of the HIV-1 env gene in 30 late-stage and 6 early-stage subjects. We also used endpoint dilution PCR followed by cloning of env genes to create pseudotyped virus to explore the link between genotypic predictions and phenotypic assessment of coreceptor usage. We found out that the most stringently sequence-based calls of X4 variants (Geno2Pheno false-positive rate [FPR] of ≤2%) formed distinct lineages within the viral population, and these were detected in 24 of 30 late-stage samples (80%), which was significantly higher than what has been seen previously by using other approaches. Non-X4 lineages were not skewed toward lower FPR scores in X4-containing populations. Phenotypic assays showed that variants with an intermediate FPR (2 to 20%) could be either X4/dual-tropic or R5 variants, although the X4 variants made up only about 25% of the lineages with an FPR of <10%, and these variants carried a distinctive sequence change. Phylogenetic analysis of both the V1/V2 and C2/V3 regions showed evidence of recombination within but very little recombination between the X4 and R5 lineages, suggesting that these populations are genetically isolated.

Importance: Primer ID sequencing provides a novel approach to study genetic structures of viral populations. X4 variants may be more prevalent than previously reported when assessed by using next-generation sequencing (NGS) and with a greater depth of sampling than single-genome amplification (SGA). Phylogenetic analysis to identify lineages of sequences with intermediate FPR values may provide additional information for accurately predicting X4 variants by using V3 sequences. Limited recombination occurs between X4 and R5 lineages, suggesting that X4 and R5 variants are genetically isolated and may be replicating in different cell types or that X4/R5 recombinants have reduced fitness.
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http://dx.doi.org/10.1128/JVI.00441-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4984628PMC
August 2016

Diversity and Tropism of HIV-1 Rebound Virus Populations in Plasma Level After Treatment Discontinuation.

J Infect Dis 2016 08 30;214(3):403-7. Epub 2016 Apr 30.

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill.

Human immunodeficiency virus-infected people discontinuing therapy experience a rebound in the virus level (hereafter, "rebound virus") from a persistent reservoir. We examined 10 samples from patients in AIDS Clinical Trials Group study A5068 with rebound virus, using single-genome amplification and Primer ID deep sequencing, to assess env genetic diversity of the virus population. Most rebound-virus populations showed significant diversity. All env examined required high levels of CD4 for entry, consistent with selection of replication in CD4(+) T cells. These results indicate that most people discontinuing therapy release a diverse population of virus and that this released virus has entry features of virus selected for replication in CD4(+) T cells, rather than in myeloid cells.
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http://dx.doi.org/10.1093/infdis/jiw172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4936648PMC
August 2016

R5 Macrophage-Tropic HIV-1 in the Male Genital Tract.

J Virol 2015 Oct 29;89(20):10688-92. Epub 2015 Jul 29.

Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

The entry tropism of HIV-1 Env proteins from virus isolated from the blood and genital tract of five men with compartmentalized lineages was determined. The Env proteins isolated from the genital tract of subject C018 were macrophage-tropic proteins, while the remaining cloned env genes encoded R5 T cell-tropic proteins. The detection of a macrophage-tropic lineage of HIV-1 within the male genital tract strongly suggests that evolution of macrophage-tropic viruses can occur in anatomically isolated sites outside the central nervous system.
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http://dx.doi.org/10.1128/JVI.01842-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4580182PMC
October 2015

Primer ID Validates Template Sampling Depth and Greatly Reduces the Error Rate of Next-Generation Sequencing of HIV-1 Genomic RNA Populations.

J Virol 2015 Aug 3;89(16):8540-55. Epub 2015 Jun 3.

UNC Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

Unlabelled: Validating the sampling depth and reducing sequencing errors are critical for studies of viral populations using next-generation sequencing (NGS). We previously described the use of Primer ID to tag each viral RNA template with a block of degenerate nucleotides in the cDNA primer. We now show that low-abundance Primer IDs (offspring Primer IDs) are generated due to PCR/sequencing errors. These artifactual Primer IDs can be removed using a cutoff model for the number of reads required to make a template consensus sequence. We have modeled the fraction of sequences lost due to Primer ID resampling. For a typical sequencing run, less than 10% of the raw reads are lost to offspring Primer ID filtering and resampling. The remaining raw reads are used to correct for PCR resampling and sequencing errors. We also demonstrate that Primer ID reveals bias intrinsic to PCR, especially at low template input or utilization. cDNA synthesis and PCR convert ca. 20% of RNA templates into recoverable sequences, and 30-fold sequence coverage recovers most of these template sequences. We have directly measured the residual error rate to be around 1 in 10,000 nucleotides. We use this error rate and the Poisson distribution to define the cutoff to identify preexisting drug resistance mutations at low abundance in an HIV-infected subject. Collectively, these studies show that >90% of the raw sequence reads can be used to validate template sampling depth and to dramatically reduce the error rate in assessing a genetically diverse viral population using NGS.

Importance: Although next-generation sequencing (NGS) has revolutionized sequencing strategies, it suffers from serious limitations in defining sequence heterogeneity in a genetically diverse population, such as HIV-1 due to PCR resampling and PCR/sequencing errors. The Primer ID approach reveals the true sampling depth and greatly reduces errors. Knowing the sampling depth allows the construction of a model of how to maximize the recovery of sequences from input templates and to reduce resampling of the Primer ID so that appropriate multiplexing can be included in the experimental design. With the defined sampling depth and measured error rate, we are able to assign cutoffs for the accurate detection of minority variants in viral populations. This approach allows the power of NGS to be realized without having to guess about sampling depth or to ignore the problem of PCR resampling, while also being able to correct most of the errors in the data set.
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http://dx.doi.org/10.1128/JVI.00522-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524263PMC
August 2015

Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain.

AIDS Res Hum Retroviruses 2015 Jun 2;31(6):658-68. Epub 2015 Apr 2.

4Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina.

Sequencing of a bulk polymerase chain reaction (PCR) product to identify drug resistance mutations informs antiretroviral therapy selection but has limited sensitivity for minority variants. Alternatively, deep sequencing is capable of detecting minority variants but is subject to sequencing errors and PCR resampling due to low input templates. We screened for resistance mutations among 184 HIV-1-infected, therapy-naive subjects using the 454 sequencing platform to sequence two amplicons spanning HIV-1 reverse transcriptase codons 34-245. Samples from 19 subjects were also analyzed using the MiSeq sequencing platform for comparison. Errors and PCR resampling were addressed by tagging each HIV-1 RNA template copy (i.e., cDNA) with a unique sequence tag (Primer ID), allowing a consensus sequence to be constructed for each original template from resampled sequences. In control reactions, Primer ID reduced 454 and MiSeq errors from 71 to 2.6 and from 24 to 1.2 errors/10,000 nucleotides, respectively. MiSeq also allowed accurate sequencing of codon 65, an important drug resistance position embedded in a homopolymeric run that is poorly resolved by the 454 platform. Excluding homopolymeric positions, 14% of subjects had evidence of ≥1 resistance mutation among Primer ID consensus sequences, compared to 2.7% by bulk population sequencing. When calls were restricted to mutations that appeared twice among consensus sequence populations, 6% of subjects had detectable resistance mutations. The use of Primer ID revealed 5-15% template utilization on average, limiting the depth of deep sequencing sampling and revealing sampling variation due to low template utilization. Primer ID addresses important limitations of deep sequencing and produces less biased estimates of low-level resistance mutations in the viral population.
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http://dx.doi.org/10.1089/AID.2014.0031DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4458739PMC
June 2015

Total daily pill burden in HIV-infected patients in the southern United States.

AIDS Patient Care STDS 2014 Jun;28(6):311-7

1 Center for Infectious Diseases, School of Medicine, UNC-Chapel Hill , Chapel Hill, North Carolina.

The need for antiretroviral therapy coupled with treatment of chronic co-morbidities places HIV-infected patients at risk for polypharmacy. However, few studies have described overall pill burden among HIV-infected patients. HIV-infected outpatients of the UNC Infectious Diseases Clinic were enrolled in this cross-sectional study. Subjects were contacted prior to a scheduled appointment and asked to bring all their medications to the visit. Daily total pill burden and medication type were recorded. 151 subjects were recruited: 76% male, 58% African American, 97% receiving antiretrovirals (ARVs). Median age was 48 (IRQ: 42-54) years. The median number of medications per subject was 8 (IQR: 6-11), and the median individual daily pill burden was 8 pills (IQR: 5-15): 3 pills (range: 2-5) for ARVs and 6 (range: 3-12.5) pills for non-ARVs. Duration of ART (per 2 years increase) and more than 3 co-morbidities was significantly associated with high pill burden (over 10 pills per day) with adjusted OR of 2.09 (95% CI, 1.14-3.84) and 8.04 (95% CI, 2.30-28.15), respectively. As patients with HIV age, strategies to reduce pill burden and number of medications will become increasingly critical to maintaining adherence, preventing medication errors, and serious drug-drug interactions.
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http://dx.doi.org/10.1089/apc.2014.0010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4180528PMC
June 2014

Monitoring HIV drug resistance using early warning indicators in China: results from a pilot survey conducted in 2008.

Clin Infect Dis 2012 May;54 Suppl 4:S300-2

Division of Treatment and Care, National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.

Robust programmatic monitoring of factors associated with the emergence of human immunodeficiency virus (HIV) drug resistance is an essential component of antiretroviral therapy (ART) program evaluation and treatment optimization. China piloted World Health Organization HIV drug resistance early warning indicators to assess the feasibility and usefulness of results. Overall, early warning indicator monitoring showed high levels of appropriate ART prescribing, low rates of loss to follow-up 12 months after ART initiation, and high rates of retention of first-line ART at 12 months. On-time drug pick-up, which may signal treatment interruptions, was identified as a challenge. HIV drug resistance early warning indicator monitoring provides a valuable assessment of ART service delivery, and its application will be scaled up throughout China.
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http://dx.doi.org/10.1093/cid/cir1018DOI Listing
May 2012

Effect of earlier initiation of antiretroviral treatment and increased treatment coverage on HIV-related mortality in China: a national observational cohort study.

Lancet Infect Dis 2011 Jul;11(7):516-24

Division of Treatment and Care, National Centre for AIDS/STD Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing, China.

Background: Overall HIV mortality rates in China have not been reported. In this analysis we assess overall mortality in treatment-eligible adults with HIV and attempt to identify risk factors for HIV-related mortality.

Methods: We used data from the national HIV epidemiology and treatment databases to identify individuals aged 15 years or older with HIV who were eligible for highly active antiretroviral therapy between 1985 and 2009. Mortality rates were calculated in terms of person-years, with risk factors determined by Cox proportional hazard regression. Treatment coverage was calculated as the proportion of time that patients who were eligible for treatment received treatment, with risk factors for not receiving treatment identified by use of logistic regression.

Findings: Of 323,252 people reported as having HIV in China by the end of 2009, 145,484 (45%) were identified as treatment-eligible and included in this analysis. Median CD4 count was 201 cells per μL (IQR 71-315) at HIV diagnosis and 194 cells per μL (73-293) when first declared eligible for treatment. Overall mortality decreased from 39·3 per 100 person-years in 2002 to 14·2 per 100 person-years in 2009, with treatment coverage concomitantly increasing from almost zero to 63·4%. By 2009, mortality was higher and treatment coverage lower in injecting drug users (15·9 deaths per 100 person-years; 42·7% coverage) and those infected sexually (17·5 deaths per 100 person-years; 61·7% coverage), compared with those infected through plasma donation or blood transfusion (6·7 deaths per 100 person-years; 80·2% coverage). The two strongest risk factors for HIV-related mortality were not receiving highly active antiretroviral therapy (adjusted hazard ratio 4·35, 95% CI 4·10-4·62) and having a CD4 count of less than 50 cells per μL when first declared eligible for treatment (7·92, 7·33-8.57).

Interpretation: An urgent need exists for earlier HIV diagnosis and better access to treatment for injecting drug users and patients infected with HIV sexually, especially before they become severely immunosuppressed.

Funding: The National Centre for AIDS/STD Control and Prevention of the Chinese Centre for Disease Control and Prevention.
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http://dx.doi.org/10.1016/S1473-3099(11)70097-4DOI Listing
July 2011

Recent key advances in human immunodeficiency virus medicine and implications for China.

AIDS Res Ther 2010 May 26;7:12. Epub 2010 May 26.

School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA.

In this article we summarize several recent major developments in human immunodeficiency virus treatment, prevention, outcome, and social policy change. Updated international guidelines endorse more aggressive treatment strategies and safer antiretroviral drugs. New antiretroviral options are being tested. Important lessons were learned in the areas of human immunodeficiency virus vaccines and microbicide gels from clinical studies, and additional trials in prevention, especially pre-exposure prophylaxis, are nearing completion. Insight into the role of the virus in the pathogenesis of diseases traditionally thought to be unrelated to acquired immunodeficiency syndrome has become a driving force for earlier and universal therapy. Lastly, we review important achievements of and future challenges facing China as she steps into her eighth year of the National Free Antiretroviral Treatment Program.
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http://dx.doi.org/10.1186/1742-6405-7-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2890503PMC
May 2010

Hepatitis B and hepatitis C seroprevalence in children receiving antiretroviral therapy for human immunodeficiency virus-1 infection in China, 2005-2009.

J Acquir Immune Defic Syndr 2010 Jun;54(2):191-6

Division of Treatment and Care, National Center for AIDS/STI Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China.

Background: Coinfection of hepatitis B virus (HBV) or hepatitis C virus (HCV) may compromise pediatric antiretroviral therapy (ART) in China. In this study, we evaluated the seroprevalence of HBV and HCV in children receiving ART and associated factors.

Methods: Patients were selected from HIV-1-infected children under age 16 enrolled in China National Pediatric ART Cohort since July 2005. Medical assessments, hepatitis B surface antigen (HBsAg), and anti-HCV antibody serologies, and transaminase levels were obtained for analysis.

Results: A total of 53 of 1082 children tested were HBsAg seropositive [4.9%; 95% confidence interval (CI): 3.6% to 6.2%], and 90 of 938 children tested were anti-HCV antibody positive (9.6%; 95% CI: 7.7% to 11.5%). No other serologic assays were performed for HBV detection. Age was associated with HBV coinfection in univariate analysis; older children were more likely to be HBsAg positive. Multivariate analysis revealed that children infected with HIV through transfusion of contaminated blood or blood products were more likely to be anti-HCV antibody positive than those infected with HIV through other routes (adjusted odds ratio = 6.2; 95% CI: 3.3% to 11.7%).

Conclusions: The high prevalence of HBV and HCV coinfection in HIV-infected children in China receiving ART demands routine screening for viral hepatitis coinfection, intensive prevention of childhood HBV and HCV transmission, and modification of the management of pediatric HIV infection.
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http://dx.doi.org/10.1097/QAI.0b013e3181c99226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877757PMC
June 2010