HIV Type 1 Intra-Subtype Superinfection Results in Increased Viral Load: a Case Report
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HIV Type 1 Intra-Subtype Superinfection Results in Increased Viral Load: A Case Report Agnes Nordquist [email protected] under the direction of Melissa Norstr¨om,PhD Candidate Department of Laboratory Medicine Karolinska Institutet Research Academy for Young Scientists July 11, 2012 Abstract Human immunodeficiency virus type 1 (HIV-1) superinfection has implications for our un- derstanding of HIV pathogenesis, transmission and vaccine development. In this study, the suspicion of superinfection in a treatment na¨ıve HIV-1 infected patient, followed from early infection up to seven years, was investigated in detail. Sequences from the viral population were obtained from the gag p24 region by single genome amplification. Bioinformatic anal- yses were performed on a dataset including sequences from ten different time points during the course of the HIV-1 infection. The findings indicate that an HIV-1 intra-subtype B super- infection occurred in this subject around one and a half year post infection. Most probably this resulted in an increase of viral load and loss of viral control. The study highlights the importance of HIV-1 superinfection and clinical consequences, which must be considered for future vaccine design. Contents 1 Introduction 4 1.1 Classification . 5 1.2 The virion . 6 1.3 The replication cycle . 7 1.4 The course of HIV-1 infection . 7 1.5 Human leukocyte antigen . 10 1.6 Superinfection . 10 2 Materials and methods 11 2.1 Patient characteristics . 11 2.2 Experimental procedures . 12 2.2.1 Viral RNA extraction . 12 2.2.2 cDNA synthesis . 12 2.2.3 Single genome amplification . 13 2.3 Bioinformatic analyses . 14 2.3.1 Editing and alignment of sequences . 14 2.3.2 Subtyping and excluding the possibility of contamination . 14 2.3.3 Excluding duplicate sequences and blasting . 15 2.3.4 Phylogenetic tree construction for all time points . 15 3 Results 16 3.1 HIV-1 disease course . 16 3.2 Experimental results . 17 3.3 Results from bioinformatic analyses . 18 3.3.1 Contamination was excluded and subtype determined as clade B . 18 1 3.3.2 Genetic variation results . 18 3.3.3 Evolutionary relationship between patient sequences . 20 4 Discussion 21 5 Acknowledgements 25 2 List of abbreviations AIDS - Acquired immune deficiency syndrome CD4 count - CD4+ T cell levels cDNA - Complimentary DNA HIV - Human immunodeficiency virus HLA - Human leukocyte antigen IFN - Interferon IL - Interleukin MHC - Major histocompatibility complex MSM - Men who have sex with men NT - Nucleotide PCR - Polymerase chain reaction RTase - Reverse transcriptase SIV - Simian immunodeficiency viruses TNF - Tumor necrosis factor VL - Viral load WPI - Weeks post infection 3 1 Introduction Human immunodeficiency virus (HIV) was in 1983 identified as the causative agent of, what has since become, one of the most devastating infectious diseases to have emerged in recent history. The HIV-virus was recognised by two separate research groups, one led by Luc Montagnier in France and one led by Robert Gallo in the United States of America [1, 2]. The virus found was the answer to the pursuit of origin of the disease acquired immune deficiency syndrome (AIDS) that first was described in the early 1980s. However, it is believed that the HIV-virus has existed since 1910 or earlier, infecting humans after evolving from a simian immunodeficiency virus (SIV) by multiple cross-species transmissions from other primate species in Africa [3]. HIV is a blood-borne pathogen. The virus spreads foremost through sexual contact, contaminated injection equipment, blood transfusions and mother-to-child during pregnancy, delivery or breastfeeding [4]. According to UNAIDS, the estimated total of people living with HIV today is 34 million, distributed across the globe (Figure 1) [5]. Figure 1: HIV-1 distribution worldwide. The HIV-1 distribution worldwide 2010 according to UNAIDS, World Health Organization [5]. 4 1.1 Classification Two HIV virus types are distinguished; HIV-1 and HIV-2. The more virulent and aggressive HIV-1 is the prevailing type in most countries while HIV-2 is endemic in West Africa. HIV is one of the fastest evolving organisms, enabling the emerge of recombinant forms of HIV from the currently present genetically distinct groups and subtypes of HIV [4]. This is a result of the error-prone viral reverse transcriptase, coupled with a high viral replication rate [6]. HIV-1 is divided in three groups: group M (for major/main) and the two minor groups N and O [7]. HIV-1 group M is spread globally. The dispersion of HIV-1 group M has led to the ascendancy of different group M lineages in different geographic areas (Figure 2). These group M lineages are currently classified in nine subtypes (A-D, F-H, J, K) and additional 40 recombinant forms (CRFs). The recombinant forms were generated when multiple subtypes infected habitants in the same geographic area [7, 8]. Figure 2: The HIV-1 global prevalence and subtype distribution. This figure shows the global HIV-1 group M subtype distribution (2004 - 2007) and the HIV-1 prevalence (2009) for each country. The subtype distribution is presented in larger regions [4]. 5 1.2 The virion The HIV virus is a lentivirus, i.e. a retrovirus with a long incubation period. The HIV virus particles are spherical with a diameter of approximately 100nm [9]. The HIV-virion has a nucleocapsid with an RNA nucleoprotein core. The virus is surrounded by a membrane derived from the host-cell containing virally encoded envelope glycoproteins (gp120 and gp41) in addition to host-cell proteins. The HIV-virion also holds protease and the enzymes reverse transcriptase and integrase. These are all necessary for viral replication. The viral particle is covered by a lipid bilayer derived from the host-cell and studded with the viral glycoproteins gp41 and gp120 (Figure 3). The HIV genome, like other retroviruses, consists of genes such as env, gag and pol. In HIV-1 the env gene codes for the precursor envelope glycoprotein (gp160). Gag encodes for the matrix (p17), capsid (p24) and nucleocapsid (p6, p7) proteins. The pol gene encodes for the viral enzymes protease, reverse transcriptase and integrase [10]. Figure 3: The HIV-1 virion. This is a schematic illustration of the HIV-1 virion [11]. 6 1.3 The replication cycle The viral replication begins with the virus envelope glycoprotein gp120 binding to a CD4 receptor and a co-receptor in the host-cell membrane, causing a change in configuration enabling the gp41 glycoprotein envelope glycoprotein to mediate fusion of the viral envelope with the membrane of the host-cell, allowing the viral genome and viral proteins entry to the cell (Figure 4). In the cytoplasm of the host-cell the viral RNA is transcribed to cDNA by reverse transcriptase. The cDNA is then transported in to the nucleus where the enzyme integrase incorporates it to the DNA of the host-cell. In a process called transcription the viral DNA is read, and the code is then used in the making of viral proteins, partially possible thanks to the enzyme integrase that cleaves proteins to their component viral peptides. The viral peptides and proteins, along with new viral RNA, forms new, immature, HIV-virions. The host-cell dies within a few days of being infected of HIV. The cell dies due to one of the following mechanisms; direct killing as a result of the virions binding to cell-surface receptors, the increased susceptibility to apoptosis or killing by cytotoxic CD8 T cells [3]. The HIV virion chiefly infects CD4+ T cells and consequently the immune system. HIV-virions infect macrophages and dendritic cells as well, as these cells express CD4 receptors just like CD4+ T cells [9]. 1.4 The course of HIV-1 infection The pathogenesis of an HIV infection depends on the virus, e.g. the level of virulence and quantity of viruses, and the host, e.g. age, genetic differences, immune responses and en- vironmental factors. Furthermore coinfection with other microbes can affect the rate and severity of disease progression [13]. However, in average the disease progression in treatment na¨ıve HIV-1 infected individuals is 10 years (Figure 5). There are exceptions with individu- als that can control the virus better, thus delay the disease progression. Likewise, there are 7 Figure 4: The HIV-1 replicatyion cycle. Schematic illustration of the HIV-1 replication cycle summarised in seven steps [12]. 8 exceptions with individuals who progress to AIDS within two to three years after infection [14]. The acute phase of HIV infection (primary) is followed by an outbreak of viraemia. For some individuals this outbreak is accompanied by flu-like symptoms. The amount of CD4+ T cells decrease rapidly [15]. However, the viraemia is reduced and peripheral CD4+ T cells are recovered with the increase in innate and HIV-specific immune responses. The point to which the viraemia is decreased is called the set point. The set point is predictive of disease progression [16]. Subsequently, the infection enters its asymptomatic, chronic, phase. During which, the HIV-specific immunity may partially control the virus. The infection can persist due to constantly emerging escape variants of the virus and the integration of HIV provirus into the cellular DNA. The chronic phase is accompanied by a gradual loss in CD4+ T cells and defect in responsiveness of several immune functions [13]. The HIV-infection progresses to AIDS when the massive reduction in the number of CD4+ T cells has lead to the number cells/mm3 becoming lower than 200. At this point the immune system is so weak that oppor- tunistic infections, e.g. due to microbes such as Candida, Toxoplasma, Herpes infections or rare cancer types such as Kaposi's Sarcoma, are observed.