Viral Replication Dynamics Research Articles

Analysis of IAV Replication and Co-infection Dynamics by a Versatile RNA Viral Genome Labeling Method

Genome delivery to the proper cellular compartment for transcription and replication is a primary goal of viruses. However, methods for analyzing viral genome localization and differentiating genomes with high identity are lacking, making it difficult to investigate entry-related processes and co-examine heterogeneous RNA viral populations. Here, we present an RNA labeling approach for single-cell analysis of RNA viral replication and co-infection dynamics insitu, which uses the versatility of padlock probes. We applied this method to identify influenza A virus (IAV) infections in cells and lung tissue with single-nucleotide specificity and to classify entry and replication stages by gene segment localization. Extending the classification strategy to co-infections of IAVs with single-nucleotide variations, we found that the dependence on intracellular trafficking places a time restriction on secondary co-infections necessary for genome reassortment. Altogether, these data demonstrate how RNA viral genome labeling can help dissect entry and co-infections.

Transmission Bottleneck Size Estimation from Pathogen Deep-Sequencing Data, with an Application to Human Influenza A Virus.

ABSTRACTThe bottleneck governing infectious disease transmission describes the size of the pathogen population transferred from the donor to the recipient host. Accurate quantification of the bottleneck size is particularly important for rapidly evolving pathogens such as influenza virus, as narrow bottlenecks reduce the amount of transferred viral genetic diversity and, thus, may decrease the rate of viral adaptation. Previous studies have estimated bottleneck sizes governing viral transmission by using statistical analyses of variants identified in pathogen sequencing data. These analyses, however, did not account for variant calling thresholds and stochastic viral replication dynamics within recipient hosts. Because these factors can skew bottleneck size estimates, we introduce a new method for inferring bottleneck sizes that accounts for these factors. Through the use of a simulated data set, we first show that our method, based on beta-binomial sampling, accurately recovers transmission bottleneck sizes, whereas other methods fail to do so. We then apply our method to a data set of influenza A virus (IAV) infections for which viral deep-sequencing data from transmission pairs are available. We find that the IAV transmission bottleneck size estimates in this study are highly variable across transmission pairs, while the mean bottleneck size of 196 virions is consistent with a previous estimate for this data set. Furthermore, regression analysis shows a positive association between estimated bottleneck size and donor infection severity, as measured by temperature. These results support findings from experimental transmission studies showing that bottleneck sizes across transmission events can be variable and influenced in part by epidemiological factors.IMPORTANCE The transmission bottleneck size describes the size of the pathogen population transferred from the donor to the recipient host and may affect the rate of pathogen adaptation within host populations. Recent advances in sequencing technology have enabled bottleneck size estimation from pathogen genetic data, although there is not yet a consistency in the statistical methods used. Here, we introduce a new approach to infer the bottleneck size that accounts for variant identification protocols and noise during pathogen replication. We show that failing to account for these factors leads to an underestimation of bottleneck sizes. We apply this method to an existing data set of human influenza virus infections, showing that transmission is governed by a loose, but highly variable, transmission bottleneck whose size is positively associated with the severity of infection of the donor. Beyond advancing our understanding of influenza virus transmission, we hope that this work will provide a standardized statistical approach for bottleneck size estimation for viral pathogens.

Suppression of chikungunya virus replication and differential innate responses of human peripheral blood mononuclear cells during co-infection with dengue virus.

Dengue and chikungunya are viral diseases transmitted to humans by infected Aedes spp. mosquitoes. With an estimated 390 million infected people per year dengue virus (DENV) currently causes the most prevalent arboviral disease. During the last decade chikungunya virus (CHIKV) has caused large outbreaks and has expanded its territory causing millions of cases in Asia, Africa and America. The viruses share a common mosquito vector and during the acute phase cause similar flu-like symptoms that can proceed to more severe or debilitating symptoms. The growing overlap in the geographical distribution of these mosquito-borne infections has led to an upsurge in reported cases of DENV/CHIKV co-infections. Unfortunately, at present we have little understanding of consequences of the co-infections to the human host. The overall aim of this study was to define viral replication dynamics and the innate immune signature involved in concurrent DENV and CHIKV infections in human peripheral blood mononuclear cells (PBMCs). We demonstrate that concomitant infection resulted in a significant reduction of CHIKV progeny and moderate enhancement of DENV production. Remarkably, the inhibitory effect of DENV on CHIKV infection occurred independently of DENV replication. Furthermore, changes in type I IFN, IL-6, IL-8, TNF-α, MCP-1 and IP-10 production were observed during concomitant infections. Notably, co-infections led to a significant increase in the levels of TNF-α and IL-6, cytokines that are widely considered to play a crucial role in the early pathogenesis of both viral diseases. In conclusion, our study reveals the interplay of DENV/CHIKV during concomitant infection and provides a framework to investigate viral interaction during co-infections.

Interpatient mutational spectrum of human coronavirus-OC43 revealed by illumina sequencing.

Human coronaviruses (HCoV) are RNA viruses that cause respiratory tract infections with viral replication of limited duration. The host and viral population heterogeneity could influence clinical phenotypes. Employing long RT‐PCR with Illumina sequencing, we quantified the gene mutation load at 0.5% mutation frequency for the 4529 bp‐domain spanning the Spike gene (4086 bp) of HCoV‐OC43 in four upper respiratory clinical specimens obtained during acute illness. There were a total of 121 mutations for all four HCoV samples with the average number of mutations at 30.3 ± 10.2, which is significantly higher than that expected from the Illumina sequencing error rate. There were two mutation peaks, one at the 5′ end and the other near position 1 550 in the S1 subunit. Two coronavirus samples were genotype B and two were genotype D, clustering with HCoV‐OC43 strain AY391777 in neighbor‐joining tree phylogenetic analysis. Nonsynonymous mutations were 76.1 ± 14% of mutation load. Although lower than other RNA viruses such as hepatitis C virus, HCoV‐OC43 did exhibit quasi‐species. The rate of nonsynonymous mutations was higher in the HCoV‐OC43 isolates than in hepatitis C (HCV) virus genotype 1a isolates analyzed for comparison in this study. These characteristics of HCoV‐OC43 may affect viral replication dynamics, receptor binding, antigenicity, evolution, transmission, and clinical illness.

Hematopoietic Stem Cell Infected with HTLV-1 Functions As a Viral Reservoir In Vivo

▪Human T-cell leukemia virus type 1 (HTLV-1) is a causative agent of a malignant disease of peripheral CD4+ T cells called adult T-cell leukemia-lymphoma (ATL) and several inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although major target of HTLV-1 is CD4+ T cells, other hematopoietic cells such as CD8+ T cells and monocytes are also infected with HTLV-1. Since the receptors of HTLV-1 are glucose transporter 1 and neuropilin 1, which are found on various cell surfaces, it is possible that HTLV-1 infects various hematopoietic cells and hematopoietic stem cells (HSCs). However, the previous studies could not detect HTLV-1 in HSCs. To assess the distribution of infected cells and expression of viral genes in various tissues, a nonhuman primate model, Japanese macaques (JMs) infected with simian T-cell leukemia virus type 1 (STLV-1) was utilized in this study. STLV-1 is a close relative of HTLV-1, and the dynamics of viral replication and proliferation of infected cells are very similar to each other. Indeed, STLV-1 caused malignant lymphomas in STLV-1 infected monkeys. Therefore, STLV-1 infected JMs are good models of HTLV-1 carriers. Using this model, we first analyzed transcription level of two viral genes, tax and STLV-1 bZIP factor, in multiple tissues, and found that tax was highly expressed in bone marrow compared to other tissues. Since Tax is a potent activator of viral transcription, this result suggested that viral replication occurred in bone marrow. To evaluate which cells express Tax in bone marrow, we performed flow cytometric analysis of bone marrow mononuclear cells from STLV-1 infected monkeys, and found that not only CD4+ T cells but also non T cells (CD3 negative cells) expressed Tax. To determine whether hematopoietic stem cells are infected with HTLV-1, we next performed highthroughput sequencing of HTLV-1 integration sites in multiple cell lineages using a next generation sequencer. Since HTLV-1 is randomly integrated into genome of infected cells, and each infected cell can proliferate clonally, we can interpret that cells sharing same integration sites are derived from the same precursor cell. Blood samples from patients with HAM/TSP were separated into five cell types: CD4+ T cells, CD8+ T cells, B cells, monocytes, and neutrophils. Integration sites of HTLV-1 provirus were analyzed by next generation sequencing using different chips for each cell type in order to avoid cross contamination between the samples. The results showed that some of these cells have same integration sites between different cell types. More than 40 percent of HTLV-1 infected B cells, monocytes and neutrophils had shared the same integration sites, suggesting that HSCs in the HAM/TSP patients were infected with HTLV-1. Importantly, most HTLV-1 infected CD4+ T cells and CD8+ T cells had independent integration sites, while some of them shared the same integration sites with non T cell subsets. These results implied that most T-cell clones were generated by de novo infection in the periphery, but a part of infected T cells were derived from infected HSCs. To assess whether HTLV-1 infected HSCs persist in vivo, we again analyzed integration sites in neutrophils from the same HAM/TSP patients after 1 year. About 50 percent of integration sites in HTLV-1 infected neutrophils were detected after 1 year. Considering short lifespan of neutrophils, this result suggests that HTLV-1 infected HSCs could be maintained for at least 1 year in vivo. To visualize HTLV-1 infection in myeloid cells, we performed immunofluorescence staining of neutrophils from HAM/TSP patients. Tax and myeloperoxidase were detected in neutrophils. Finally, we evaluated the significance of infected monocytes in viral transmission. We isolated monocytes from HAM/TSP patients and co-cultured with JET WT35 cell, which is a subline of Jurkat containing a fluorescent reporter of viral infection. The result demonstrated that monocyte from HAM/TSP patients could be a source of infective HTLV-1. In conclusion, our findings suggest that HSCs infected with HTLV-1 survive for long time in vivo, and could be reservoirs of the virus. Since ATL is a difficult disease to cure, further studies are required to understand the nature of HTLV-1 infection. DisclosuresNo relevant conflicts of interest to declare.

Large Variations in HIV-1 Viral Load Explained by Shifting-Mosaic Metapopulation Dynamics

The viral population of HIV-1, like many pathogens that cause systemic infection, is structured and differentiated within the body. The dynamics of cellular immune trafficking through the blood and within compartments of the body has also received wide attention. Despite these advances, mathematical models, which are widely used to interpret and predict viral and immune dynamics in infection, typically treat the infected host as a well-mixed homogeneous environment. Here, we present mathematical, analytical, and computational results that demonstrate that consideration of the spatial structure of the viral population within the host radically alters predictions of previous models. We study the dynamics of virus replication and cytotoxic T lymphocytes (CTLs) within a metapopulation of spatially segregated patches, representing T cell areas connected by circulating blood and lymph. The dynamics of the system depend critically on the interaction between CTLs and infected cells at the within-patch level. We show that for a wide range of parameters, the system admits an unexpected outcome called the shifting-mosaic steady state. In this state, the whole body’s viral population is stable over time, but the equilibrium results from an underlying, highly dynamic process of local infection and clearance within T-cell centers. Notably, and in contrast to previous models, this new model can explain the large differences in set-point viral load (SPVL) observed between patients and their distribution, as well as the relatively low proportion of cells infected at any one time, and alters the predicted determinants of viral load variation.

Zika viral dynamics and shedding in rhesus and cynomolgus macaques.

Infection with Zika virus has been associated with serious neurological complications and fetal abnormalities. However, the dynamics of viral infection, replication and shedding are poorly understood. Here we show that both rhesus and cynomolgus macaques are highly susceptible to infection by lineages of Zika virus that are closely related to, or are currently circulating in, the Americas. After subcutaneous viral inoculation, viral RNA was detected in blood plasma as early as 1 d after infection. Viral RNA was also detected in saliva, urine, cerebrospinal fluid (CSF) and semen, but transiently in vaginal secretions. Although viral RNA during primary infection was cleared from blood plasma and urine within 10 d, viral RNA was detectable in saliva and seminal fluids until the end of the study, 3 weeks after the resolution of viremia in the blood. The control of primary Zika virus infection in the blood was correlated with rapid innate and adaptive immune responses. We also identified Zika RNA in tissues, including the brain and male and female reproductive tissues, during early and late stages of infection. Re-infection of six animals 45 d after primary infection with a heterologous strain resulted in complete protection, which suggests that primary Zika virus infection elicits protective immunity. Early invasion of Zika virus into the nervous system of healthy animals and the extent and duration of shedding in saliva and semen underscore possible concern for additional neurologic complications and nonarthropod-mediated transmission in humans.

Compartmentalized dynamics of cytomegalovirus replication in treated congenital infection

BackgroundCytomegalovirus (CMV) is the most prevalent congenital infection in developed countries. A significant number of infected infants develop long-term neurodevelopmental and hearing impairment irrespective of whether disease is detectable at birth. Studies of viral load and replication dynamics have informed the treatment of CMV in adult populations but no similar data exist in neonates. ObjectivesTo study CMV virus kinetics in different body fluids of babies treated for congenital infection. Study designCMV virus load was sequentially analyzed in blood, urine and saliva in 17 babies treated for symptomatic congenital CMV infection. ResultsVirus was detectable in the urine and saliva of all babies at baseline but in only 15/17 in blood. At the end of 6 weeks of antiviral treatment CMV remained detectable in 9/14 blood samples, 9/12 urine samples and 4/7 salivary swabs. Median half-life (T1/2) of virus decline in blood was 2.4 days (IQR 1.9–3.3) and basic reproductive number (Ro) was 2.3. Although T1/2 values were similar in urine and saliva to those observed in blood, virus dynamics differed both during and after treatment. ConclusionsT1/2 and Ro in blood in this group of neonates were similar to values derived from studies of immunocompromised adults. The persistent viremia observed in treated neonates cannot therefore be adequately explained by the virus dynamics early in treatment. The different dynamics exhibited in blood and urine suggests that studying changes in distinct body compartments may assist in further understanding long-term manifestations of disease.

Modelling Virus and Antibody Dynamics during Dengue Virus Infection Suggests a Role for Antibody in Virus Clearance.

Dengue is an infection of increasing global importance, yet uncertainty remains regarding critical aspects of its virology, immunology and epidemiology. One unanswered question is how infection is controlled and cleared during a dengue infection. Antibody is thought to play a role, but little past work has examined the kinetics of both virus and antibody during natural infections. We present data on multiple virus and antibody titres measurements recorded sequentially during infection from 53 Vietnamese dengue patients. We fit mechanistic mathematical models of the dynamics of viral replication and the host immune response to these data. These models fit the data well. The model with antibody removing virus fits the data best, but with a role suggested for ADCC or other infected cell clearance mechanisms. Our analysis therefore shows that the observed viral and antibody kinetics are consistent with antibody playing a key role in controlling viral replication. This work gives quantitative insight into the relationship between antibody levels and the efficiency of viral clearance. It will inform the future development of mechanistic models of how vaccines and antivirals might modify the course of natural dengue infection.

Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells.

Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-016-7542-4) contains supplementary material, which is available to authorized users.

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Human nasal epithelial cells derived from multiple subjects exhibit differential responses to H3N2 influenza virus infection in vitro

Fiat Luc: Bioluminescence Imaging Reveals In Vivo Viral Replication Dynamics

Animal models are invaluable in studying viral replication in vivo, the pathogenesis of viral infection, the host immune response, and the efficacy of antiviral interventions. Despite their utility, animal models have been constrained by the inability to monitor viral replication dynamics in real time. Cohorts of animals are infected and euthanized, and viral load or immune responses are measured in predetermined tissues. This produces static snapshots of replication only at specific times and specific sites within the animal. Moreover, inherent animal-to-animal variability introduces significant confounding effects, resulting in studies that require hundreds of animals to acquire statistical significance. The power of these traditional approaches can be complemented, and many of these limitations overcome, by using in vivo bioluminescence imaging (BLI) (Fig 1). BLI detects light produced by luciferase enzymes. A growing list of viruses have been engineered to express luciferase and exploit this technology (Table 1), enabling rapid measures of viral load over time (i.e., longitudinal measurements), tissue distribution, interhost transmission, and the impact of therapeutic treatments in animal models. Open in a separate window Fig 1 Illuminating viral replication dynamics with bioluminescence imaging. An influenza reporter virus encoding NanoLuc (NLuc) is used to demonstrate how real-time noninvasive bioluminescence imaging is used (A) to study replication and dissemination in the same animal over time; (B) to measure the impact of antiviral treatment with the neuraminidase inhibitor Tamiflu (oseltamivir) on viral load and tissue distribution; and (C) in multimodal imaging of infected mice mock treated or treated with Tamiflu, in which viral load and distribution was measured by BLI and host inflammatory responses were measured by PET/CT with the radiotracer [18F]-2-deoxy-2-fluoro-D-glucose. Viral load and inflammation are colored from purple to red, representing low to high levels. Construction of the reporter virus and its use in mice are detailed in Tran, et al. [6].

Single-Cell and Single-Cycle Analysis of HIV-1 Replication

The dynamics of the late stages of the HIV-1 life cycle are poorly documented. Viral replication dynamics are typically measured in populations of infected cells, but asynchrony that is introduced during the early steps of HIV-1 replication complicates the measurement of the progression of subsequent steps and can mask replication dynamics and their variation in individual infected cells. We established microscopy-based methods to dynamically measure HIV-1-encoded reporter gene and antiviral gene expression in individual infected cells. We coupled these measurements with conventional analyses to quantify delays in the HIV-1 replication cycle imposed by the biphasic nature of HIV-1 gene expression and by the assembly-inhibiting property of the matrix domain of Gag. We further related the dynamics of restriction factor (APOBEC3G) removal to the dynamics of HIV-1 replication in individual cells. These studies provide a timeline for key events in the HIV-1 replication cycle, and reveal that the interval between the onset of early and late HIV-1 gene expression is only ~3h, but matrix causes a ~6–12h delay in the generation of extracellular virions. Interestingly, matrix delays particle assembly to a time at which APOBEC3G has largely been removed from the cell. Thus, a need to prepare infected cells to be efficient producers of infectious HIV-1 may provide an impetus for programmed delays in HIV-1 virion genesis. Our findings also emphasize the significant heterogeneity in the length of the HIV-1 replication cycle in homogenous cell populations and suggest that a typical infected cell generates new virions for only a few hours at the end of a 48h lifespan. Therefore, small changes in the lifespan of infected cells might have a large effect on viral yield in a single cycle and the overall clinical course in infected individuals.

Dynamic models of viral replication and latency.

Purpose of reviewHIV targets primary CD4+ T cells. The virus depends on the physiological state of its target cells for efficient replication, and, in turn, viral infection perturbs the cellular state significantly. Identifying the virus–host interactions that drive these dynamic changes is important for a better understanding of viral pathogenesis and persistence. The present review focuses on experimental and computational approaches to study the dynamics of viral replication and latency.Recent findingsIt was recently shown that only a fraction of the inducible latently infected reservoirs are successfully induced upon stimulation in ex-vivo models while additional rounds of stimulation make allowance for reactivation of more latently infected cells. This highlights the potential role of treatment duration and timing as important factors for successful reactivation of latently infected cells. The dynamics of HIV productive infection and latency have been investigated using transcriptome and proteome data. The cellular activation state has shown to be a major determinant of viral reactivation success. Mathematical models of latency have been used to explore the dynamics of the latent viral reservoir decay.SummaryTiming is an important component of biological interactions. Temporal analyses covering aspects of viral life cycle are essential for gathering a comprehensive picture of HIV interaction with the host cell and untangling the complexity of latency. Understanding the dynamic changes tipping the balance between success and failure of HIV particle production might be key to eradicate the viral reservoir.

Mechanistic dynamics of Hepatitis C virus replication in single liver cells

Infection with hepatitis C virus (HCV) causes chronic liver diseases. HCV-related liver damage is the main reason for liver transplantations in the western world. Spatial resolution is an aspect that has not yet been appreciated in current modeling simulations despite strong biological evidence that suggests intracellular spatial dependence is a crucial factor in the process the virus uses to replicate its RNA genome. HCV replication is believed to occur in specialized compartments within virus-infected cells, termed replication complexes, which are derived from altered regions (called the membranous web) of the endoplasmic reticulum (ER – the ER is a central structure of each cell). Trafficking of both replication complexes, and their components, is likely a dynamic process occurring in three dimensions that is both difficult to capture experimentally and conceptually visualize. Therefore, we are developing a spatially- resolved biophysical model of HCV replication dynamics in single liver cells. We used data derived from 3D confocal microscopy of HCV-infected human hepatoma cells labeled for the ER membrane in order to reconstruct 3D geometries of single hepatocytes using NeuRA2. On top of these geometries, we developed a model using (surface) partial differential equation of viral RNA replication dynamics with particular emphasis upon RNA movement, viral protein production, cleavage and movement, and viral RNA replication within the membranous web. The arising (s)pde s on the ER surface are solved using the simulation platform UG4 within a Finite Volume framework combined with multigrid techniques. Our approach is based on two columns which are intended to grow together in the middle run: On the one hand side, we are doing parameter estimations of single components of viral replication based on the Gauss-Newton algorithm in order for extracting e.g. the diffusion constant of basic viral proteins on the surface of the ER using experimental FRAP time series. On the other hand side, we are developing the model which mimics the interplay of all important component of virus replication, e.g. viral RNA and various states of viral proteins. The estimated parameters are entering the model step by step. Therefore, the presented work is a practical application of the powerful modeling/simulation tool UG4 and its multigrid techiques for the case of a huge number of degrees of freedom (DoFs) already and demonstrates the excellent usability (and scalability) of UG4 in the context of modern biophysical research.

Influenza A virus utilizes a suboptimal Kozak sequence to fine-tune virus replication and host response

The segment-specific non-coding regions (NCRs) of influenza A virus RNA genome play important roles in controlling viral RNA transcription, replication and genome packaging. In this report, we present, for the first time to our knowledge, a full view of the segment-specific NCRs of all influenza A viruses by bioinformatics analysis. Our systematic functional analysis revealed that the eight segment-specific NCRs identified could differentially regulate viral RNA synthesis and protein expression at both transcription and translation levels. Interestingly, a highly conserved suboptimal nucleotide at -3 position of the Kozak sequence, which downregulated protein expression at the translation level, was only present in the segment-specific NCR of PB1. By reverse genetics, we demonstrate that recombinant viruses with an optimized Kozak sequence at the -3 position in PB1 resulted in a significant multiple-cycle replication reduction that was independent of PB1-F2 expression. Our detailed dynamic analysis of virus infection revealed that the mutant virus displays slightly altered dynamics from the wild-type virus on both viral RNA synthesis and protein production. Furthermore, we demonstrated that the level of PB1 expression is involved in regulating type I IFN production. Together, these data reveal a novel strategy exploited by influenza A virus to fine-tune virus replication dynamics and host antiviral response through regulating PB1 protein expression.

Construction and characterization of a recombinant invertebrate iridovirus

Chilo iridescent virus (CIV), officially named Insect iridescent virus 6 (IIV6), is the type species of the genus Iridovirus (family Iridoviridae). In this paper we constructed a recombinant CIV, encoding the green fluorescent protein (GFP). This recombinant can be used to investigate viral replication dynamics. We showed that homologous recombination is a valid method to make CIV gene knockouts and to insert foreign genes. The CIV 157L gene, putatively encoding a non-functional inhibitor of apoptosis (IAP), was chosen as target for foreign gene insertion. The gfp open reading frame preceded by the viral mcp promoter was inserted into the 157L locus by homologous recombination in Anthonomus grandis BRL-AG-3A cells. Recombinant virus (rCIV-Δ157L-gfp) was purified by successive rounds of plaque purification. All plaques produced by the purified recombinant virus emitted green fluorescence due to the presence of GFP. One-step growth curves for recombinant and wild-type CIV were similar and the recombinant was fully infectious in vivo. Hence, CIV157L can be inactivated without altering the replication kinetics of the virus. Consequently, the CIV 157L locus can be used as a site for insertion of foreign DNA, e.g. to modify viral properties for insect biocontrol.

Disentangling the HIV-1 human protein interaction networks and their implications in the dynamics of viral replication and pathogenesis

Results Sixteen HIV-1 proteins interacted with 2545 human proteins, of which 3668 (34.53%) were direct and 6340 (65.46%), regulatory interactions. Seventy seven different types of interactions with T cell surface glycoprotein CD4 isoform 1 precursor (276/10008) and DNA dC->dU editing enzyme APOBEC-3G(109) being the top two interacting proteins. The top interacting HIV-1 proteins were tat, p14 (28%) followed by gp120 and nef p27. Functional annotation returned 312 clusters with highest enrichment scores of 30.71 for positive regulation of apoptosis (p value 7.9E-32). Toll like receptor and the Jak sTAT signaling pathways were most crucial to host response. LOCATE identified 491(10.7%) nuclear proteins, 419(9.15%) cytoplasmic, 295 (6.44%) membrane and 190(4.15%) extracellular proteins.

Pathogenic features associated with increased virulence upon Simian immunodeficiency virus cross-species transmission from natural hosts.

While simian immunodeficiency viruses (SIVs) are generally nonpathogenic in their natural hosts, dramatic increases in pathogenicity may occur upon cross-species transmission to new hosts. Deciphering the drivers of these increases in virulence is of major interest for understanding the emergence of new human immunodeficiency viruses (HIVs). We transmitted SIVsab from the sabaeus species of African green monkeys (AGMs) to pigtailed macaques (PTMs). High acute viral replication occurred in all SIVsab-infected PTMs, yet the outcome of chronic infection was highly variable, ranging from rapid progression to controlled infection, which was independent of the dynamics of acute viral replication, CD4(+) T cell depletion, or preinfection levels of microbial translocation. Infection of seven PTMs with plasma collected at necropsy from a rapid-progressor PTM was consistently highly pathogenic, with high acute and chronic viral replication, massive depletion of memory CD4(+) T cells, and disease progression in all PTMs. The plasma inoculum used for the serial passage did not contain adventitious bacterial or viral contaminants. Single-genome amplification showed that this inoculum was significantly more homogenous than the inoculum directly derived from AGMs, pointing to a strain selection in PTMs. In spite of similar peak plasma viral loads between the monkeys in the two passages, immune activation/inflammation levels dramatically increased in PTMs infected with the passaged virus. These results suggest that strain selection and a massive cytokine storm are major factors behind increased pathogenicity of SIV upon serial passage and adaptation of SIVs to new hosts following cross-species transmission. We report here that upon cross-species transmission and serial passage of SIVsab from its natural host, the sabaeus African green monkey (AGM), to a new host, the pigtailed macaque (PTM), viral adaptation and increased pathogenicity involve strain selection and a massive cytokine storm. These results permit the design of strategies aimed at preventing cross-species transmission from natural hosts of SIVs to humans in areas of endemicity. Furthermore, our study describes a new animal model for SIV infection. As the outcomes of SIVsab infection in PTMs, African green monkeys, and rhesus macaques are different, the use of these systems enables comparative studies between pathogenic, nonpathogenic, and elite-controlled infections, to gain insight into the mechanisms of SIV immunodeficiency and comorbidities.

Role of BK virus infection in end‐stage renal disease patients waiting for kidney transplantation – viral replication dynamics from pre‐ to post‐transplant

We report the prevalence of BK virus (BKV) infection before renal transplantation and the dynamics of BKV viremia from pre- to post-transplantation. We assessed 60 kidney transplanted patients from a single cohort in Italy, treated with identical immunosuppressive therapy, for BK viremia at pre-transplantation, 12 h, and three and six months post-transplantation. Polymerase chain reaction showed that the prevalence of plasma BKV replication--considered a marker of infection--was 20% in pre-transplant patients. All pre-transplant-positive patients remained positive post-transplant, whereas the majority of pre-transplant-negative patients remained negative. Viremia dynamics classification revealed three clusters of patients: Cluster A++, pre-transplant-positive patients (20%) who tested positive at least once post-transplant; Cluster B-+, pre-transplant-negative patients (28%) who tested positive at least once post-transplant; and Cluster C- -, pre-transplant-negative patients (52%) who remained negative throughout. These clusters presented significant differences related to the prevalence of substantially positive patients with high plasma viral load (>10(3) copies/mL) in cluster A, but not in donors' or grafts' characteristics. We suggest that pre-transplant viral status should be considered as an additional risk factor for post-transplant BKV replication. Therefore, pre-transplant BKV infection screening in kidney transplant patients should be performed for improving planning of personalized immunosuppressant schemes and specific post-transplant surveillance.