Evolution of an Eurasian Avian-like Influenza Virus in Na?ve and Vaccinated Pigs

Influenza viruses are characterized by an ability to cross species boundaries and evade host immunity, sometimes with devastating consequences. The 2009 pandemic of H1N1 influenza A virus highlights the importance of pigs in influenza emergence, particularly as intermediate hosts by which avian viruses adapt to mammals before emerging in humans. Although segment reassortment has commonly been associated with influenza emergence, an expanded host-range is also likely to be associated with the accumulation of specific beneficial point mutations. To better understand the mechanisms that shape the genetic diversity of avian-like viruses in pigs, we studied the evolutionary dynamics of an Eurasian Avian-like swine influenza virus (EA-SIV) in naïve and vaccinated pigs linked by natural transmission. We analyzed multiple clones of the hemagglutinin 1 (HA1) gene derived from consecutive daily viral populations. Strikingly, we observed both transient and fixed changes in the consensus sequence along the transmission chain. Hence, the mutational spectrum of intra-host EA-SIV populations is highly dynamic and allele fixation can occur with extreme rapidity. In addition, mutations that could potentially alter host-range and antigenicity were transmitted between animals and mixed infections were commonplace, even in vaccinated pigs. Finally, we repeatedly detected distinct stop codons in virus samples from co-housed pigs, suggesting that they persisted within hosts and were transmitted among them. This implies that mutations that reduce viral fitness in one host, but which could lead to fitness benefits in a novel host, can circulate at low frequencies.     

Evidence of Early B-Cell Dysregulation in Simian Immunodeficiency Virus Infection: Rapid Depletion of Na?ve and Memory B-Cell Subsets with Delayed Reconstitution of the Na?ve B-Cell Population

Despite eliciting a robust antibody response in humans, several studies in human immunodeficiency virus (HIV)-infected patients have demonstrated the presence of B-cell deficiencies during the chronic stage of infection. While several explanations for the HIV-induced B-cell deficit have been proposed, a clear mechanistic understanding of this loss of B-cell functionality is not known. This study utilizes simian immunodeficiency virus (SIV) infection of rhesus macaques to assess B-cell population dynamics beginning at the acute phase and continuing through the chronic phase of infection. Flow cytometric assessment demonstrated a significant early depletion of both naïve and memory B-cell subsets in the peripheral blood, with differential kinetics for recovery of these populations. Furthermore, the altered numbers of naïve and memory B-cell subsets in these animals corresponded with increased B-cell activation and altered proliferation profiles during the acute phase of infection. Finally, all animals produced high titers of antibody, demonstrating that the measurement of virus-specific antibody responses was not an accurate reflection of alterations in the B-cell compartment. These data indicate that dynamic B-cell population changes in SIV-infected macaques arise very early after infection at the precise time when an effective adaptive immune response is needed.Effective B-cell responses result in the generation of memory B-cell populations which are able to proliferate and produce antibodies that can control primary and secondary insults by microbial pathogens (2). Impaired maturation and timing of B-cell-mediated immune responses result in the production of ineffective antibodies, which are unable to control infection and may result in the persistence of the pathogen (36). Although human immunodeficiency virus (HIV) infection generally elicits high-titer antibodies, virus-specific titers do not correlate with delayed clinical progression, suggesting that antibodies produced during HIV infection are not sufficient to provide long-term viral control (6). Ineffective antibody production in the context of HIV infection could be a result of numerous T-cell and B-cell abnormalities induced either directly or indirectly through infection. B-cell perturbations, characterized during chronic infection, include hypergammaglobulinemia (11, 31), a diminished in vitro response to mitogenic stimulation (10, 37), diminished antibody responses to vaccination (15, 23), and loss of memory B-cell subsets (3, 10, 37). It is highly likely that these B-cell abnormalities are linked with the inability of HIV-infected individuals to form effective antibody responses to HIV and opportunistic pathogens.B-cell perturbations during acute HIV infection may lead to dysfunctions observed during chronic infection. Despite numerous reports that hypothesized that B-cell phenotypic and functional abnormalities arise due to the effects of chronic infection, a limited number of acute infection studies have provided evidence that B-cell dysfunctions may be initiated much earlier. Studies by De Milito et al. and others have reported a decrease in CD27⁺ B cells associated with chronic HIV infection (3, 4, 10-12, 15, 30, 31, 36-38, 40). The reduction of this population may explain the diminished antibody responses to non-HIV antigens present in HIV-infected individuals. However, the mechanism for this loss of memory B cells during chronic infection is unclear. One possibility is that B-cell losses are related to reduced T-cell numbers. In a study by Titanji et al., a strong correlation between the number of CD4 T cells and the percentage of memory B cells was reported in chronic HIV infection (37). Conversely, others have reported that no correlation was found between CD4 numbers and memory B-cell numbers (3, 10). Interestingly, reductions in percentages of B cells, increased expression of Fas on B cells, increased total plasma IgG levels, a decreased percentage of IgM memory B cells, and decreased B-cell responses to antigenic stimulation have been shown to occur within 6 months of HIV infection (36, 37). Disruption of germinal centers in the gut during acute HIV infection may also compromise the humoral immune response (20). While these studies provide insight into virus-induced changes in the B-cell compartment during infection, it is difficult to ascertain precisely when these changes occur, due to limitations in sample size and numbers during this early period of infection. The conflicting reports reflect the high amount of variability present in human HIV infection and illuminate the need for a model to study B-cell populations in which experimental parameters can be more rigorously controlled. An understanding of the effects of HIV on the B-cell population during this critical early phase of infection is needed to determine how the initial interactions between virus and host immune system set the stage for long-term disease progression in the infected host. The simian immunodeficiency virus (SIV)/macaque model provides a system in which to ask these questions.Studies in SIV-infected macaques have demonstrated that the number of total B (CD20⁺) cells in the periphery decreases dramatically during the acute phase of infection (13, 24). The loss of these cells coincides with a similar depletion of peripheral CD4 T cells and is associated with primary viremia. Interestingly, the loss of total B cells is greater in magnitude than the loss of CD4⁺ T cells (24). In order to understand how these cells are being depleted, it is necessary to characterize B-cell subsets during SIV infection in the macaque. The present study was designed to assess phenotypic changes in B-cell numbers during the acute phase of SIV infection, both in the total B-cell population as well as in B-cell subsets. Our results identified early, rapid changes in B-cell subsets that were not apparent in analysis of the total B-cell population. Specifically, we identified a significant depletion from the periphery of both the naïve (CD20⁺ CD27⁻) and memory (CD20⁺ CD27⁺) B-cell populations during acute infection and increased total B-cell population activation that may be related to ineffective antibody production commonly associated with SIV infection. Furthermore, the data demonstrate that measurement of envelope-specific antibody responses was not a sensitive reflection of SIV effects on B-cell subsets. These data provide novel information about the timing and dynamics of phenotypic changes in the B-cell compartment during SIV infection that may be associated with functional changes observed later in chronic infection. These results can be used to tailor therapeutic treatments designed to preserve the B-cell compartment early in SIV/HIV infection.     

Dynamics of Onchocerca volvulus Microfilarial Densities after Ivermectin Treatment in an Ivermectin-na?ve and a Multiply Treated Population from Cameroon

Background/Objective

Ivermectin has been the keystone of onchocerciasis control for the last 25 years. Sub-optimal responses to the drug have been reported in Ghanaian communities under long-term treatment. We assessed, in two Cameroonian foci, whether the microfilaricidal and/or embryostatic effects of ivermectin on Onchocerca volvulus have been altered after several years of drug pressure.

Methods

We compared the dynamics of O. volvulus skin microfilarial densities after ivermectin treatment in two cohorts with contrasting exposure to this drug: one received repeated treatment for 13 years whereas the other had no history of large-scale treatments (referred to as controls). Microfilarial densities were assessed 15, 80 and 180 days after ivermectin in 122 multiply treated and 127 ivermectin-naïve individuals. Comparisons were adjusted for individual factors related to microfilarial density: age and number of nodules.

Findings

Two weeks post ivermectin, microfilarial density dropped equally (98% reduction) in the ivermectin-naïve and multiply treated groups. Between 15 and 180 days post ivermectin, the proportion of individuals with skin microfilariae doubled (from 30.8% to 67.8%) in controls and quadrupled (from 19.8% to 76.9%) in multiply treated individuals but the mean densities remained low in both sites. In fact, between 15 and 80 days, the repopulation rate was significantly higher in the multiply treated individuals than in the controls but no such difference was demonstrated when extending the follow-up to 180 days. The repopulation rate by microfilariae was associated with host factors: negatively with age and positively with the number of nodules.

Conclusion

These observations may indicate that the worms from the multi-treated area recover mf productivity earlier but would be less productive than the worms from the ivermectin-naïve area between 80 and 180 days after ivermectin. Moreover, they do not support the operation of a strong cumulative effect of repeated treatments on the fecundity of female worms as previously described.     

Molecular Dynamics in Protein—Single Stranded DNA Complexes. Two Distinct Nucleoside Mobilities in Poly(deoxythymidylic acid)—poly-L-lysine Complexes

Abstract

Stoichiometric amounts of poly-L-lysine were added to site-specifically spin labeled single stranded nucleic acids and the resulting complexes analyzed by electron spin resonance spectroscopy (ESR). The nucleic acids were spin labeled to different extents and with labels of varying tether length. The ESR data are used to determine nucleoside dynamics and some structural features in these complexes. It is concluded that two distinct base mobilities exist in the complexes; one set is characterized by a mean correlation time τR = 2 ns, and the other one by a τR ≤ 50 ns. A model is proposed which suggests that a poly-L-lys single stranded nucleic acid complex consists of low mobility segments flanked by more mobile bases. An interesting feature of the proposed model is its applicability to explain ESR data of single strand binding protein-spin labeled nucleic acid complexes, which can also be interpreted in terms of two distinct nucleoside mobility states. It is hypothesized that this phenomenon could be of biological significance for the release of protein ligands from a protein-nucleic acid complex.     

Effect of Porcine Epidemic Diarrhea Virus Infectious Doses on Infection Outcomes in Na?ve Conventional Neonatal and Weaned Pigs

Porcine epidemic diarrhea virus (PEDV) was identified in the United States (U.S.) swine population for the first time in April 2013 and rapidly spread nationwide. However, no information has been published regarding the minimum infectious dose (MID) of PEDV in different pig models. The main objective of this study was to determine the oral minimum infectious dose of PEDV in naïve conventional neonatal piglets and weaned pigs. A U.S. virulent PEDV prototype isolate (USA/IN19338/2013) with known infectious titer was serially ten-fold diluted in virus-negative cell culture medium. Dilutions with theoretical infectious titers from 560 to 0.0056 TCID₅₀/ml together with a medium control were orogastrically inoculated (10ml/pig) into 7 groups of 5-day-old neonatal pigs (n = 4 per group) and 7 groups of 21-day-old weaned pigs (n = 6 per group). In 5-day-old pigs, 10ml of inoculum having titers 560–0.056 TCID₅₀/ml, corresponding to polymerase chain reaction (PCR) cycle threshold (Ct) values 24.2–37.6, resulted in 100% infection in each group; 10ml of inoculum with titer 0.0056 TCID₅₀/ml (Ct>45) caused infection in 25% of the inoculated pigs. In 21-day-old pigs, 10ml of inoculum with titers 560–5.6 TCID₅₀/ml (Ct 24.2–31.4) resulted in 100% infection in each group while 10ml of inoculum with titers 0.56–0.0056 TCID₅₀/ml (Ct values 35.3 –>45) did not establish infection in any pigs under study conditions as determined by clinical signs, PCR, histopathology, immunohistochemistry, and antibody response. These data reveal that PEDV infectious dose is age-dependent with a significantly lower MID for neonatal pigs compared to weaned pigs. This information should be taken into consideration when interpreting clinical relevance of PEDV PCR results and when designing a PEDV bioassay model. The observation of such a low MID in neonates also emphasizes the importance of strict biosecurity and thorough cleaning/disinfection on sow farms.     

Evidence for Persistence of Ectromelia Virus in Inbred Mice,Recrudescence Following Immunosuppression and Transmission to Na?ve Mice

Orthopoxviruses (OPV), including variola, vaccinia, monkeypox, cowpox and ectromelia viruses cause acute infections in their hosts. With the exception of variola virus (VARV), the etiological agent of smallpox, other OPV have been reported to persist in a variety of animal species following natural or experimental infection. Despite the implications and significance for the ecology and epidemiology of diseases these viruses cause, those reports have never been thoroughly investigated. We used the mouse pathogen ectromelia virus (ECTV), the agent of mousepox and a close relative of VARV to investigate virus persistence in inbred mice. We provide evidence that ECTV causes a persistent infection in some susceptible strains of mice in which low levels of virus genomes were detected in various tissues late in infection. The bone marrow (BM) and blood appeared to be key sites of persistence. Contemporaneous with virus persistence, antiviral CD8 T cell responses were demonstrable over the entire 25-week study period, with a change in the immunodominance hierarchy evident during the first 3 weeks. Some virus-encoded host response modifiers were found to modulate virus persistence whereas host genes encoded by the NKC and MHC class I reduced the potential for persistence. When susceptible strains of mice that had apparently recovered from infection were subjected to sustained immunosuppression with cyclophosphamide (CTX), animals succumbed to mousepox with high titers of infectious virus in various organs. CTX treated index mice transmitted virus to, and caused disease in, co-housed naïve mice. The most surprising but significant finding was that immunosuppression of disease-resistant C57BL/6 mice several weeks after recovery from primary infection generated high titers of virus in multiple tissues. Resistant mice showed no evidence of a persistent infection. This is the strongest evidence that ECTV can persist in inbred mice, regardless of their resistance status.     

Is the In?Vitro Ejection of Bacteriophage DNA Quasistatic? A Bulk to Single Virus Study

Bacteriophage T5 DNA ejection is a complex process that occurs on several timescales in vitro. By using a combination of bulk and single phage measurements, we quantitatively study the three steps of the ejection—binding to the host receptor, channel-opening, and DNA release. Each step is separately addressed and its kinetics parameters evaluated. We reconstruct the bulk kinetics from the distribution of single phage events by following individual DNA molecules with unprecedented time resolution. We show that, at the single phage level, the ejection kinetics of the DNA happens by rapid transient bursts that are not correlated to any genome sequence defects. We speculate that these transient pauses are due to local phase transitions of the DNA inside the capsid. We predict that such pauses should be seen for other phages with similar DNA packing ratios.     

In Vitro Selection of Single‐Stranded DNA Aptamers that Bind Human Pro‐Urokinase

Single‐chain pro‐urokinase is an inactive proenzyme form of human urokinase (urinary plasminogen activator) with a Mr of 50,000 which is converted to the active two‐chain form by catalytic amounts of plasmin. It is used for thrombolytic therapy of acute myocardial infarction and acute ischemic stroke. We have isolated single‐stranded DNA molecules with significantly increased binding affinity for human pro‐urokinase by SELEX (systematic evolution of ligands by exponential enrichment) procedure from a pool of 10¹⁵ molecules containing 24 randomized positions which are flanked by defined regions. ssDNA from this library was hybridized with helper «fixture», thus allowing the central random chain to fold into complex three‐dimentional shapes. Sequencing data from pro‐urokinase aptamers obtained after 12 selection cycles displayed a highly conserved 12–14 base region.     

Host genes involved in the replication of single-stranded DNA phage ?K

Summary Using various replication mutants of E. coli, the host genes that participate in the replication of some K12-specific single-stranded DNA phages have been determined. Functional products of dnaE,-F,-G and -Z genes are required for the multiplication of K, whereas dnaA,-B,-C(D), H,-I and -P are dispensable for viral replication. In contrast with polB, recA, B, C, or xth functions, host rep activity is essential for K. At the restrictive temperature, the yield of K was markedly reduced in the ligts7 mutant and partially decreased in a polA ^ts strain. The phage K is thus less dependent on the host cells than X174 and A which require additionally the dnaB,-C(D) and -H functions. Replication of phage St-1 depends on dnaG and -Z gene products, but not on dnaP function. Although not much affected in polA ^ts host, growth of St-1 was significantly diminished in dnaF or ligts7 mutants.     

Nature of φX174 Linear DNA from a DNA Ligase-Defective Host

Linear DNAs have been prepared from phiX phage and from phiX RF II (double-stranded circular form of phiX DNA, formed during infection and nicked in one or both strands) molecules derived from infection at the restrictive temperature of Escherichia coli ts7, a host mutant with a temperature-sensitive DNA ligase activity. The linear DNA from these phages can be circularized by annealing with fragments of phiX RF DNA produced by the Haemophilus influenzae restriction nuclease. The circularization experiment indicated that the site of breakage of the linear phage DNAs is not unique nor confined to a particular region of the genome. These linear DNAs were less than 0.1% as infective as circular phage DNA. The linear, positive strand of late RF II DNA, however, is uniquely nicked in the region of the phiX genome corresponding to cistron A. Although a low level of infectivity is associated with the linear DNA derived from late RF II, this infectivity appears to be a result of the association of linear positive and linear negative strands during the infectivity assay.     

Porcine Epidemic Diarrhea Virus RNA Present in Commercial Spray-Dried Porcine Plasma Is Not Infectious to Na?ve Pigs

Porcine epidemic diarrhea virus emerged in North America in April 2013 and has since been identified in 30 U.S. States, Canada and Mexico. The rapid spread of PEDV has raised concerns about the role of feed and particularly pork-by-product components such as spray-dried porcine plasma (SDPP) in PEDV transmission. The aim of this study was to determine the infectivity of PEDV RNA present in commercial SDPP. Specifically, 40 3-week-old PEDV naïve pigs were randomly assigned to one of five treatment groups. At day post inoculation (dpi) 0, NEG-CONTROL pigs were sham-inoculated, PEDV-CONTROL pigs received cell culture propagated PEDV, and SDPP-CONTROL pigs were switched to a diet with 5% SDPP containing 5.1±0.1 log₁₀ PEDV RNA copies/g. To evaluate a potential positive effect of anti-PEDV antibodies in SDPP on PEDV challenge, four days prior to PEDV challenge the pigs in the SDPP-PEDV group were switched to and remained on a 5% SDPP diet through dpi 28. Another group, EGG-PEDV, was orally administered a commercial egg-derived liquid PEDV globulin product from dpi -4 through 6. All PEDV-CONTROL pigs began shedding PEDV in feces by dpi 3 and seroconverted between dpi 7 and 14, whereas pigs in NEG-CONTROL and SDPP-CONTROL groups remained PEDV RNA negative and did not seroconvert to PEDV for the study duration. This indicates no evidence of infectivity of the PEDV RNA in the SDPP lot utilized. Furthermore, under the study conditions SDPP or egg-derived liquid PEDV globulin addition did not significantly alter PEDV-shedding or overall disease course after experimental challenge.     

Interaction of the ΦHSIC Virus with Its Host: Lysogeny or Pseudolysogeny?

The marine phage ΦHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified as Listonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at −80^° C and were lost. When HSIC was reinfected with phage ΦHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (10¹¹ ml⁻¹) in the absence of inoculating phage and yet reached culture densities of nearly 10⁹ ml⁻¹. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the ΦHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that ΦHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.     

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Na?ve Patients with Non-Valvular Atrial Fibrillation

Genotype-guided warfarin dosing have been proposed to improve patient’s management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naïve patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR<2.0 or >3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01178034","term_id":"NCT01178034"}}NCT01178034     

Abacavir-Reactive Memory T Cells Are Present in Drug Na?ve Individuals

BackgroundFifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.MethodsTo determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.ResultsAbacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.ConclusionsWe propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.     

Transmission or Within-Host Dynamics Driving Pulses of Zoonotic Viruses in Reservoir–Host Populations

Progress in combatting zoonoses that emerge from wildlife is often constrained by limited knowledge of the biology of pathogens within reservoir hosts. We focus on the host–pathogen dynamics of four emerging viruses associated with bats: Hendra, Nipah, Ebola, and Marburg viruses. Spillover of bat infections to humans and domestic animals often coincides with pulses of viral excretion within bat populations, but the mechanisms driving such pulses are unclear. Three hypotheses dominate current research on these emerging bat infections. First, pulses of viral excretion could reflect seasonal epidemic cycles driven by natural variations in population densities and contact rates among hosts. If lifelong immunity follows recovery, viruses may disappear locally but persist globally through migration; in either case, new outbreaks occur once births replenish the susceptible pool. Second, epidemic cycles could be the result of waning immunity within bats, allowing local circulation of viruses through oscillating herd immunity. Third, pulses could be generated by episodic shedding from persistently infected bats through a combination of physiological and ecological factors. The three scenarios can yield similar patterns in epidemiological surveys, but strategies to predict or manage spillover risk resulting from each scenario will be different. We outline an agenda for research on viruses emerging from bats that would allow for differentiation among the scenarios and inform development of evidence-based interventions to limit threats to human and animal health. These concepts and methods are applicable to a wide range of pathogens that affect humans, domestic animals, and wildlife.