An exploratory algorithm to identify intra-host recombinant viral sequences

Hepatitis C virus (HCV) causes chronic infection in up to 50% to 80% of infected individuals. Hypervariable region 1 (HVR1) variability is frequently studied to gain an insight into the mechanisms of HCV adaptation during chronic infection, but the changes to and persistence of HCV subpopulations during intrahost evolution are poorly understood. In this study, we used ultradeep pyrosequencing (UDPS) to map the viral heterogeneity of a single patient over 9.6 years of chronic HCV genotype 4a infection. Informed error correction of the raw UDPS data was performed using a temporally matched clonal data set. The resultant data set reported the detection of low-frequency recombinants throughout the study period, implying that recombination is an active mechanism through which HCV can explore novel sequence space. The data indicate that polyvirus infection of hepatocytes has occurred but that the fitness quotients of recombinant daughter virions are too low for the daughter virions to compete against the parental genomes. The subpopulations of parental genomes contributing to the recombination events highlighted a dynamic virome where subpopulations of variants are in competition. In addition, we provide direct evidence that demonstrates the growth of subdominant populations to dominance in the absence of a detectable humoral response. IMPORTANCEAnalysis of ultradeep pyrosequencing data sets derived from virus amplicons frequently relies on software tools that are not optimized for amplicon analysis, assume random incorporation of sequencing errors, and are focused on achieving higher specificity at the expense of sensitivity. Such analysis is further complicated by the presence of hypervariable regions. In this study, we made use of a temporally matched reference sequence data set to inform error correction algorithms. Using this methodology, we were able to (i) detect multiple instances of hepatitis C virus intrasubtype recombination at the E1/E2 junction (a phenomenon rarely reported in the literature) and (ii) interrogate the longitudinal quasispecies complexity of the virome. Parallel to the UDPS, isolation of IgG-bound virions was found to coincide with the collapse of specific viral subpopulations.

Section: Discussionsupporting

confidence: 76%

Analysis of the Evolution and Structure of a Complex Intrahost Viral Population in Chronic Hepatitis C Virus Mapped by Ultradeep Pyrosequencing

Palmer

Dimitrova

Skums

et al. 2014

“…Recombination analysis was carried out for sequence populations in each tissue and in each macaque using three methods, as previously described (51): (i) GARD (52), (ii) an algorithm that uses split decomposition networks in combination with the "phitest" (53,54) implemented in Splitstree4 software (55) calibrated specifically to identify likely recombinants within intrahost viral populations (53,56,57), and (iii) Recombine (58). Recombinant sequences were also identified using bootscanning, implemented in Simplot (59) and RDP (60), with observed trends similar to those obtained with the algorithms listed above (data not shown).…”

Section: Methodsmentioning

confidence: 99%

Evolution of Neuroadaptation in the Periphery and Purifying Selection in the Brain Contribute to Compartmentalization of Simian Immunodeficiency Virus (SIV) in the Brains of Rhesus Macaques with SIV-Associated Encephalitis

Rife

Nolan

Lamers³

et al. 2016

Self Cite

The emergence of a distinct subpopulation of human or simian immunodeficiency virus (HIV/SIV) sequences within the brain (compartmentalization) during infection is hypothesized to be linked to AIDS-related central nervous system (CNS) neuropathology. However, the exact evolutionary mechanism responsible for HIV/SIV brain compartmentalization has not been thoroughly investigated. Using extensive viral sampling from several different peripheral tissues and cell types and from three distinct regions within the brain from two well-characterized rhesus macaque models of the neurological complications of HIV infection (neuroAIDS), we have been able to perform in-depth evolutionary analyses that have been unattainable in HIV-infected subjects. The results indicate that, despite multiple introductions of virus into the brain over the course of infection, brain sequence compartmentalization in macaques with SIV-associated CNS neuropathology likely results from late viral entry of virus that has acquired through evolution in the periphery sufficient adaptation for the distinct microenvironment of the CNS. IMPORTANCEHIV-associated neurocognitive disorders remain prevalent among HIV type 1-infected individuals, whereas our understanding of the critical components of disease pathogenesis, such as virus evolution and adaptation, remains limited. Building upon earlier findings of specific viral subpopulations in the brain, we present novel yet fundamental results concerning the evolutionary patterns driving this phenomenon in two well-characterized animal models of neuroAIDS and provide insight into the timing of entry of virus into the brain and selective pressure associated with viral adaptation to this particular microenvironment. Such knowledge is invaluable for therapeutic strategies designed to slow or even prevent neurocognitive impairment associated with AIDS. Human immunodeficiency virus type 1 (HIV-1)-associated neurocognitive disorders (HAND) remain a persistent complication despite the success of highly active antiretroviral therapy (HAART) in prolonging the progression to AIDS in HIV-1-infected patients (1). Although the post-HAART era has witnessed a decrease in the prevalence of the most severe form of HAND, HIV-associated dementia (HAD), an increase in prevalence has been observed for the milder forms, referred to as asymptomatic neurocognitive impairment (ANI) and mild neurocognitive disorder (MND) (2, 3). Moreover, these assessments have been reported to be an underestimation of the true prevalence, based on a study in which approximately 64% of HIV-1-infected patients classified as "noncomplainers," self-reporting no symptoms of cognitive impairment, tested positive for HAND using comprehensive neurocognitive testing criteria (4). Furthermore, with the life expectancy for patients receiving HAART now projected to be greater than 70 years (5, 6), the reported prevalence estimations can only be expected to increase, whereas our understanding of the critical components of disease pathogenesis, such as t...

“…Due to the large number of insertions and deletions that are typically problematic to align, a portion of the highly variable gp120 V1 domain (SIVmm239 positions 6987 to 7035) was removed. All nef and gp120 sequences were tested for recombination using the PHI test implemented in Splitstree4 (40), as previously described (41,42), and suspected recombinants were removed prior to distance and phylogenetic analyses. All alignments are available upon request.…”

Section: Methodsmentioning

confidence: 99%

Tracking the Emergence of Host-Specific Simian Immunodeficiency Virus env and nef Populations Reveals nef Early Adaptation and Convergent Evolution in Brain of Naturally Progressing Rhesus Macaques

Lamers¹,

Nolan

Rife

et al. 2015

Self Cite

While a clear understanding of the events leading to successful establishment of host-specific viral populations and productive infection in the central nervous system (CNS) has not yet been reached, the simian immunodeficiency virus (SIV)-infected rhesus macaque provides a powerful model for the study of human immunodeficiency virus (HIV) intrahost evolution and neuropathogenesis. The evolution of the gp120 and nef genes, which encode two key proteins required for the establishment and maintenance of infection, was assessed in macaques that were intravenously inoculated with the same viral swarm and allowed to naturally progress to simian AIDS and potential SIV-associated encephalitis (SIVE). Longitudinal plasma samples and immune markers were monitored until terminal illness. Single-genome sequencing was employed to amplify full-length env through nef transcripts from plasma over time and from brain tissues at necropsy. nef sequences diverged from the founder virus faster than gp120 diverged. Host-specific sequence populations were detected in nef (ϳ92 days) before they were detected in gp120 (ϳ182 days). At necropsy, similar brain nef sequences were found in different macaques, indicating convergent evolution, while gp120 brain sequences remained largely host specific. Molecular clock and selection analyses showed weaker clock-like behavior and stronger selection pressure in nef than in gp120, with the strongest nef selection in the macaque with SIVE. Rapid nef diversification, occurring prior to gp120 diversification, indicates that early adaptation of nef in the new host is essential for successful infection. Moreover, the convergent evolution of nef sequences in the CNS suggests a significant role for nef in establishing neurotropic strains. IMPORTANCEThe SIV-infected rhesus macaque model closely resembles HIV-1 immunopathogenesis, neuropathogenesis, and disease progression in humans. Macaques were intravenously infected with identical viral swarms to investigate evolutionary patterns in the gp120 and nef genes leading to the emergence of host-specific viral populations and potentially linked to disease progression. Although each macaque exhibited unique immune profiles, macaque-specific nef sequences evolving under selection were consistently detected in plasma samples at 3 months postinfection, significantly earlier than in gp120 macaque-specific sequences. On the other hand, nef sequences in brain tissues, collected at necropsy of two animals with detectable infection in the central nervous system (CNS), revealed convergent evolution. The results not only indicate that early adaptation of nef in the new host may be essential for successful infection, but also suggest that specific nef variants may be required for SIV to efficiently invade CNS macrophages and/or enhance macrophage migration, resulting in HIV neuropathology. Aclear understanding of the events leading to the establishment of human immunodeficiency virus type 1 (HIV-1) infection in a new host, including the central nervous system (C...