Experimental Andes Virus Infection in Deer Mice: Characteristics of Infection and Clearance in a Heterologous Rodent Host

New World hantaviruses can cause hantavirus cardiopulmonary syndrome with high mortality in humans. Distinct virus species are hosted by specific rodent reservoirs, which also serve as the vectors. Although regional spillover has been documented, it is unknown whether rodent reservoirs are competent for infection by hantaviruses that are geographically separated, and known to have related, but distinct rodent reservoir hosts. We show that Andes virus (ANDV) of South America, carried by the long tailed pygmy rice rat (Oligoryzomys longicaudatus), infects and replicates in vitro and in vivo in the deer mouse (Peromyscus maniculatus), the reservoir host of Sin Nombre virus (SNV), found in North America. In experimentally infected deer mice, viral RNA was detected in the blood, lung, heart and spleen, but virus was cleared by 56 days post inoculation (dpi). All of the inoculated deer mice mounted a humoral immune response by 14 dpi, and produced measurable amounts of neutralizing antibodies by 21 dpi. An up-regulation of Ccl3, Ccl4, Ccl5, and Tgfb, a strong CD4⁺ T-cell response, and down-regulation of Il17, Il21 and Il23 occurred during infection. Infection was transient with an absence of clinical signs or histopathological changes. This is the first evidence that ANDV asymptomatically infects, and is immunogenic in deer mice, a non-natural host species of ANDV. Comparing the immune response in this model to that of the immune response in the natural hosts upon infection with their co-adapted hantaviruses may help clarify the mechanisms governing persistent infection in the natural hosts of hantaviruses.     

Association between IL28B Polymorphisms and Spontaneous Clearance of Hepatitis B Virus Infection

Background/Aims

Single-nucleotide polymorphisms (SNPs) near the interleukin 28B gene (IL28B; interferon [IFN]-λ-3) are associated with outcomes of chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection treated with peginterferon (PEG-IFN) alpha-based antiviral therapy. In this study, we investigated the influence of IL28B polymorphisms on spontaneous clearance of HBV infection in a large Korean cohort.

Methods

Between January 2007 and June 2010, a total of 208 patients with chronic HBV infection and newly diagnosed HBV-related hepatocellular carcinoma were recruited as the CC group [HBsAg(+) for >6 months, anti-HBc(+), and anti-HBs(-)]. In addition, 351 organ donors were stratified into the UE group [n = 106; HBsAg(-), anti-HBc(-), and anti-HBs(-)] or the SC group [n = 245; HBsAg(-), anti-HBc(+), and anti-HBs(+)]. The SNaPshot ddNTP Primer Extension Kit (Applied Biosystems, Foster City, CA) was used for SNP detection. Direct full sequencing of the IL28B coding region was attempted.

Results

Regardless of group, rs12979860 CC was most frequently identified (85.0% in UE, 85.9% in SC, and 93.5% in CC, respectively), whereas rs12979860 TT was not identified in any group. Similarly, rs12980275 AA and rs8099917 TT were most frequently identified (≥85%) regardless of group, whereas rs12980275 GG was identified in only one subject in the SC group. In addition, rs8099917 GG was not identified. The prevalences of CC in rs12979860, AA in rs12980275, and TT in rs8099917 were significantly higher in the CC group when compared with the UE and SC group (all P<0.05). Among 19 novel SNPs in the IL28B coding region, the proportions of 6 SNPs were significantly different among the UE, SC, and CC groups (all P<0.05).

Conclusions

The SNP upstream of IL28B that has the strongest genetic association with HCV recovery has an inverse influence on HBV recovery. Additional studies are needed to understand the mechanisms of this SNP in HBV infection.     

Experimental Myocarditis Induced in Mice by Infection with Influenza A2 Virus

A mouse model was established for the study of acute myocarditis that occurs during influenza infection. Challenge with more than 10 LD₅₀ of mouse-adapted influenza A₂ virus (H₂N₂) induced myocarditis macroscopically discernible as white, irregularly shaped lesions which were shown by histological examination to consist of necrotic myofibers surrounded by infiltrating mononuclear inflammatory cells. After challenge with 10 LD₅₀ of the virus, macroscopic myocarditis was found to advance in a progressive manner up to the 7th day, while the virus titer in the heart reached its peak on the 2nd day and began to decrease on the 5th day of infection. However, development of myocarditis was significantly suppressed in mice which were irradiated with 400 R of X-rays before infection. In addition, myocarditis did not develop in congenitally athymic nude mice. These data indicate that myocarditis was not brought about by viral action directly, but that it was mediated by some function of the host against viral in-vasion, which was abolished by X-irradiation. The data also suggest that T cells played a key role in the development of myocarditis.     

The Impact of Hepatitis A Virus Infection on Hepatitis C Virus Infection: A Competitive Exclusion Hypothesis

We address the observation that, in some cases, patients infected with the hepatitis C virus (HCV) are cleared of HCV when super-infected with the hepatitis A virus (HAV). We hypothesise that this phenomenon can be explained by the competitive exclusion principle, including the action of the immune system, and show that the inclusion of the immune system explains both the elimination of one virus and the co-existence of both infections for a certain range of parameters. We discuss the potential clinical implications of our findings.     

Acute SIV Infection in Sooty Mangabey Monkeys Is Characterized by Rapid Virus Clearance from Lymph Nodes and Absence of Productive Infection in Germinal Centers

Lymphoid tissue immunopathology is a characteristic feature of chronic HIV/SIV infection in AIDS-susceptible species, but is absent in SIV-infected natural hosts. To investigate factors contributing to this difference, we compared germinal center development and SIV RNA distribution in peripheral lymph nodes during primary SIV infection of the natural host sooty mangabey and the non-natural host pig-tailed macaque. Although SIV-infected cells were detected in the lymph node of both species at two weeks post infection, they were confined to the lymph node paracortex in immune-competent mangabeys but were seen in both the paracortex and the germinal center of SIV-infected macaques. By six weeks post infection, SIV-infected cells were no longer detected in the lymph node of sooty mangabeys. The difference in localization and rate of disappearance of SIV-infected cells between the two species was associated with trapping of cell-free virus on follicular dendritic cells and higher numbers of germinal center CD4⁺ T lymphocytes in macaques post SIV infection. Our data suggests that fundamental differences in the germinal center microenvironment prevent productive SIV infection within the lymph node germinal centers of natural hosts contributing to sustained immune competency.     

Immune-Mediated Competition in Rodent Malaria Is Most Likely Caused by Induced Changes in Innate Immune Clearance of Merozoites

Malarial infections are often genetically diverse, leading to competitive interactions between parasites. A quantitative understanding of the competition between strains is essential to understand a wide range of issues, including the evolution of virulence and drug resistance. In this study, we use dynamical-model based Bayesian inference to investigate the cause of competitive suppression of an avirulent clone of Plasmodium chabaudi (AS) by a virulent clone (AJ) in immuno-deficient and competent mice. We test whether competitive suppression is caused by clone-specific differences in one or more of the following processes: adaptive immune clearance of merozoites and parasitised red blood cells (RBCs), background loss of merozoites and parasitised RBCs, RBC age preference, RBC infection rate, burst size, and within-RBC interference. These processes were parameterised in dynamical mathematical models and fitted to experimental data. We found that just one parameter , the ratio of background loss rate of merozoites to invasion rate of mature RBCs, needed to be clone-specific to predict the data. Interestingly, was found to be the same for both clones in single-clone infections, but different between the clones in mixed infections. The size of this difference was largest in immuno-competent mice and smallest in immuno-deficient mice. This explains why competitive suppression was alleviated in immuno-deficient mice. We found that competitive suppression acts early in infection, even before the day of peak parasitaemia. These results lead us to argue that the innate immune response clearing merozoites is the most likely, but not necessarily the only, mediator of competitive interactions between virulent and avirulent clones. Moreover, in mixed infections we predict there to be an interaction between the clones and the innate immune response which induces changes in the strength of its clearance of merozoites. What this interaction is unknown, but future refinement of the model, challenged with other datasets, may lead to its discovery.     

Effects of Reticuloendotheliosis Virus Infection on Cytokine Production in SPF Chickens

Infection with reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, can result in immunosuppression and subsequent increased susceptibility to secondary infections. The effects of REV infection on expression of mRNA for cytokine genes in chickens have not been completely elucidated. In this study, using multiplex branched DNA (bDNA) technology, we identified molecular mediators that participated in the regulation of the immune response during REV infection in chickens. Cytokine and chemokine mRNA expression levels were evaluated in the peripheral blood mononuclear cells (PBMCs). Expression levels of interleukin (IL)-4, IL-10, IL-13 and tumor necrosis factor (TNF)-α were significantly up-regulated while interferon (IFN)-α, IFN-β, IFN-γ, IL-1β，IL-2, IL-3, IL-15, IL-17F, IL-18 and colony-stimulating factor (CSF)-1 were markedly decreased in PBMCs at all stages of infection. Compared with controls, REV infected chickens showed greater expression levels of IL-8 in PBMCs 21 and 28 days post infection. In addition, REV regulates host immunity as a suppressor of T cell proliferative responses. The results in this study will help us to understand the host immune response to virus pathogens.     

A Role for Ifit2 in Restricting West Nile Virus Infection in the Brain

Previous studies have demonstrated that type I interferon (IFN-I) restricts West Nile virus (WNV) replication and pathogenesis in peripheral and central nervous system (CNS) tissues. However, the in vivo role of specific antiviral genes that are induced by IFN-I against WNV infection remains less well characterized. Here, using Ifit2^−/− mice, we defined the antiviral function of the interferon-stimulated gene (ISG) Ifit2 in limiting infection and disease in vivo by a virulent North American strain of WNV. Compared to congenic wild-type controls, Ifit2^−/− mice showed enhanced WNV infection in a tissue-restricted manner, with preferential replication in the CNS of animals lacking Ifit2. Virological analysis of cultured macrophages, dendritic cells, fibroblasts, cerebellar granule cell neurons, and cortical neurons revealed cell type-specific antiviral functions of Ifit2 against WNV. In comparison, small effects of Ifit2 were observed on the induction or magnitude of innate or adaptive immune responses. Our results suggest that Ifit2 restricts WNV infection and pathogenesis in different tissues in a cell type-specific manner.     

Role of N-Linked Glycosylation of the 5-HT2A Receptor in JC Virus Infection

JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR). The 5-HT_2AR contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT_2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT_2AR-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT_2AR to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT_2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT_2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT_2AR resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT_2AR-expressing cells. These data affirm the importance of 5-HT_2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT_2AR.The initial interaction between virus and host occurs via molecular interactions of viral attachment proteins and receptors on host cells. Therefore, receptor recognition is a critical host cell determinant and may play a key regulatory role in viral pathogenesis. The polyomavirus JC virus (JCV) is a ubiquitous human pathogen (21, 25, 32) that is initially subclinical yet establishes a persistent infection in the kidney (11). In immunosuppressed individuals JCV can become reactivated, leading to infection in the central nervous system (CNS) (13-15, 20), where the virus specifically targets glial cells, including astrocytes and the myelin-producing cells, oligodendrocytes (40, 48). JCV infection and cytolytic destruction of oligodendroglia cause the fatal disease progressive multifocal leukoencephalopathy (PML) (1, 22). The most common cause of PML is associated with human immunodeficiency virus (HIV) and AIDS (10, 23). However, in recent years PML has been reported in patients receiving immunosuppressive therapies for autoimmune diseases such as Crohn''s disease (44), multiple sclerosis (MS) (24, 26, 28, 47), systemic lupus erythematosus (5, 33), and rheumatoid arthritis (5, 19, 37). The prognosis of PML is bleak, as the disease progresses rapidly and usually proves fatal within 1 year of the onset of symptoms. While current treatment options for PML are limited (23), recent studies suggest that mirtazapine, a serotonin receptor antagonist, may be capable of slowing the progression of PML (6, 27, 45, 46).JCV has a nonenveloped, icosahedral capsid that encapsidates a circular double-stranded DNA (dsDNA) genome (39). JCV attachment to cells is mediated by an N-linked glycoprotein with either α(2,3)- or α(2,6)-linked sialic acid (16, 31), suggesting that N-linked glycosylation of cellular receptors is important for JCV infection. N-linked glycosylation is a posttranslational process by which oligosaccharides are added to asparagine residues, and this modification is important for protein processing, folding, expression, and function (43). Previous studies from our laboratory revealed that the JCV also requires the serotonin 5-hydroxytryptamine_2A receptor (5-HT_2AR) to mediate JCV infection (18, 35, 38), while others report that JCV infection can occur in the absence of 5-HT_2AR (7, 8). 5-HT_2AR is a seven-transmembrane-spanning G-protein-coupled receptor that belongs to a large family of 5-HT serotonin receptors. 5-HT_2AR is abundantly expressed on cells in the brain (4), including glial cells (3), and in the kidney (4), which parallels the sites of JCV infection. N-linked glycosylation plays a key regulatory role in the function of serotonin receptors. Mutation of N-linked glycosylation sites in human 5-HT_3AR and 5-HT_5AR results in decreased expression at the plasma membrane, which is critical for receptor function (17, 34). N-linked glycosylation of murine 5-HT_3AR regulates plasma membrane targeting, ligand binding, Ca²⁺ flux, and receptor trafficking (36), suggesting that glycosylation is essential for expression and function of serotonin receptors.While previous studies have concluded that JCV utilizes an N-linked glycoprotein with α(2,3)-linked sialic acid (31) or α(2,6)-linked sialic acid (16) and 5-HT_2AR (18) to initiate infection in host cells, the mechanism(s) by which JCV engages its cellular receptors and the importance of receptor glycosylation remain unclear. 5-HT_2AR contains potential asparagine (N)-linked glycosylation sites, five of which are predicted to be expressed in the extracellular amino-terminal region, where they could be accessible to the virus (2). The goal of this study was to determine whether potential N-linked glycosylation sites expressed in 5-HT_2AR are required for JCV infection. We found that N-linked glycosylation of 5-HT_2AR is important for receptor expression but not necessary for JCV infection.     

Role that each NKG2A immunoreceptor tyrosine-based inhibitory motif plays in mediating the human CD94/NKG2A inhibitory signal

The human NKG2A chain of the CD94/NKG2A receptor contains two immunoreceptor Tyr-based inhibitory motifs (ITIMs) in its cytoplasmic tail. To determine the relative importance of membrane-distal (residues 6-11) and membrane-proximal (residues 38-43) ITIMs in mediating the inhibitory signal, we made site-directed mutants of NKG2A at the Y (Y8F, Y40F, Y8F/Y40F) and the residues two positions N-terminal (Y-2) of Y (V6A, I38A, V6A/I38A) in each motif. Wild-type (wt) and mutated NKG2A were then cotransfected with CD94 into rat basophilic leukemia 2H3 cells. Immunochemical analyses after pervanadate treatment showed that each of the mutant molecules could be phosphorylated to expected levels relative to wt NKG2A and that all the mutations significantly reduced the avidity of SH2 domain-bearing tyrosine phosphatase-1 for NKG2A. Confocal microscopy was used to determine whether SH2 domain-bearing tyrosine phosphatase-1 and CD94/NKG2A colocalized intracellularly after receptor ligation. Only the Y8F/Y40F and Y8F mutant NKG2A molecules failed to show a dramatic colocalization. In agreement with this result, the Y8F/Y40F mutant was unable to inhibit FcepsilonRI-mediated serotonin release and the Y8F mutant was relatively ineffective compared with wt NKG2A. In contrast, the Y40F mutant was 70% as effective as wt in mediating inhibition, and the Y-2 mutations did not remarkably affect inhibitory function. These results show that, like KIR, both NKG2A ITIMs are required for mediating the maximal inhibitory signal, but opposite to KIR, the membrane-distal ITIM is of primary importance rather than the membrane-proximal ITIM. This probably reflects the opposite orientation of the ITIMs in type II vs type I proteins.     

肥胖可能与病毒感染有关

随着生活水平的提高,肥胖已呈现为全球性问题.当今已将肥胖与艾滋病、癌症并称为21世纪威胁人类健康的三大疾病.而其中肥胖症则是人类健康的最大威胁.当前人们普遍认为肥胖是由于遗传、过食、运动不足、代谢异常、内分泌异常、脑疾患等因素引起.但是这些均不能完满地解释肥胖的成因.为此,美国科学家Dhurandhar等提出了一种新的解释.他们认为,肥胖可能与病毒感染有关[1].并已研究发现了第1株可引起动物肥胖的人腺病毒血清36型(adenouirus Ad-36),这是迄今所发现的第1株与肥胖有关的人腺病毒[2].证据表明,该病毒感染在动物肥胖发病中起着重要作用[2~5].是否能导致人类肥胖尚不得而知.但是,有关肥胖的病毒发现使肥胖成因的研究有了新的思考,为人类进一步了解肥胖问题的根源,以及肥胖的防治开辟了新的领域,展现出新的希望.至于病毒导致动物肥胖的具体机理以及人类肥胖是否与病毒感染有关至今尚不明确,有待深入研究.     

Local Blockade of Epithelial PDL-1 in the Airways Enhances T Cell Function and Viral Clearance during Influenza Virus Infection

In order to maintain the gas exchange function of the lung following influenza virus infection, a delicate orchestration of positive and negative regulatory pathways must be maintained to attain viral eradication while minimizing local inflammation. The programmed death receptor 1 ligand/programmed death receptor 1 (PDL-1/PD-1) pathway plays an important immunoregulatory role, particularly in the context of T cell function. Here, we have shown that influenza virus infection of primary airway epithelial cells strongly enhances PDL-1 expression and does so in an alpha interferon receptor (IFNAR) signaling-dependent manner. PD-1 is expressed primarily on effector T cells in the lung, compared to effector memory and central memory cells, and shortly after influenza virus infection, an increased number of PD-1⁺ T cells are recruited to the airways. Using in vitro cocultures of airway epithelial cells and T cells and in vivo models of influenza virus infection, we have demonstrated that blockade of airway epithelial PDL-1 improves CD8 T cell function, defined by increased production of gamma interferon (IFN-γ) and granzyme B and expression of CD107ab. Furthermore, PDL-1 blockade in the airways served to accelerate influenza virus clearance and enhance infection recovery. Our findings suggest that local manipulation of the PDL-1/PD-1 axis in the airways may represent a therapeutic alternative during acute influenza virus infection.     

Preexisting Infection with Human T-Cell Lymphotropic Virus Type 2 neither Exacerbates nor Attenuates Simian Immunodeficiency Virus SIVmac251 Infection in Macaques

Coinfection with human T-cell lymphotropic virus type 2 (HTLV-2) and human immunodeficiency virus type 1 (HIV-1) has been reported to have either a slowed disease course or to have no effect on progression to AIDS. In this study, we generated a coinfection animal model and investigated whether HTLV-2 could persistently infect macaques, induce a T-cell response, and impact simian immunodeficiency virus SIV_mac251-induced disease. We found that inoculation of irradiated HTLV-2-infected T cells into Indian rhesus macaques elicited humoral and T-cell responses to HTLV-2 antigens at both systemic and mucosal sites. Low levels of HTLV-2 provirus DNA were detected in the blood, lymphoid tissues, and gastrointestinal tracts of infected animals. Exposure of HTLV-2-infected or naïve macaques to SIV_mac251 demonstrated comparable levels of SIV_mac251 viral replication, similar rates of mucosal and peripheral CD4⁺ T-cell loss, and increased T-cell proliferation. Additionally, neither the magnitude nor the functional capacity of the SIV-specific T-cell-mediated immune response was different in HTLV-2/SIV_mac251 coinfected animals versus SIV_mac251 singly infected controls. Thus, HTLV-2 targets mucosal sites, persists, and importantly does not exacerbate SIV_mac251 infection. These data provide the impetus for the development of an attenuated HTLV-2-based vectored vaccine for HIV-1; this approach could elicit persistent mucosal immunity that may prevent HIV-1/SIV_mac251 infection.Human T-cell lymphotropic virus type 2 (HTLV-2) was discovered in 1982 and recognized as the second human retrovirus found (29). HTLV-2 is closely related to the first human retrovirus discovered, HTLV-1 (49, 50)_, a pathogenic virus that causes adult T-cell leukemia/lymphoma (ATLL) and an inflammatory neurologic disorder called HTLV-1-associated myelopathy or tropical spastic paraparesis (HAM/TSP) (22, 45).HTLV-2 is prevalent in Amerindian populations of North and South America and in Africa (57). The prevalence of HTLV-2 is generally low; however, in the past 20 years, an epidemic of HTLV-2 infection has occurred among intravenous drug users (8, 24, 54, 57). HTLV-2 establishes a lifelong infection and replicates at low levels in most infected individuals. While anecdotal cases of TSP/HAM-like neurological manifestations (1, 44) and hematopoietic diseases, such as large granular lymphoma (LGL), in HTLV-2-infected individuals have been reported (3, 37-39, 46), the extent to which HTLV-2 can induce disease in humans remains unclear. Indeed, even in the condition of immune deficiency, such as infection with human immunodeficiency virus type 1 (HIV-1), HTLV-2 coinfection has not been reported to be associated with cancer or neurological diseases. However, more studies are necessary to fully understand the role of HTLV-2 in human disease. While HTLV-1 infection has been connected with an accelerated course of disease in HIV-1 coinfected patients (2, 34), HTLV-2 has been reported to either have no effect (26) or suggested to exert a potential protective role during HIV-1 infection (12, 23). This protective role is thought to be due to a maintenance of CD4⁺ T cells, lowering immune activation, and delayed progression to AIDS (4, 5). In addition, modulation of cytokine and chemokine networks by HTLV-2 has been suggested to contribute to the control of HIV-1 infection (12, 36, 47). Since studies on the immunological interactions between HIV-1 and HTLV-2 have been performed in patients coinfected with HIV-1 and HTLV-2 in the chronic phase of HIV-1 disease, little is known about the effects of HTLV-2 infection during acute HIV-1 replication, mucosal CD4⁺ T-cell depletion, or HIV-1-specific immune responses. Furthermore, the potential protective effect of an HTLV-2 vector that would target both CD4⁺ and CD8⁺ T cells and induce a low-grade persistent infection makes HTLV-2 an interesting potential vaccine platform for an HIV-1 vaccine.Current HIV-1 vaccine strategies have focused on viral vectors delivering HIV-1 antigens. These vectors stimulate strong, systemic antigen-specific responses but are unable to protect from infection, since they generate only limited mucosal responses and do not persist. The only vaccine approach that has conferred protection in the simian immunodeficiency virus SIV_mac251 macaque model is a live attenuated virus (17), suggesting that persistent expression of viral antigens in mucosal and lymphoid tissues may be necessary. An HTLV-2 vector expressing HIV-1 antigens at mucosal sites that stimulates and maintains T-cell responses in the gut may confer protection from infection by quickly eliminating cells infected by the founder virus at the portal of entry. This study establishes that the Indian rhesus macaque model for HTLV-2 infection is a suitable model to test this hypothesis, as it demonstrates that HTLV-2 targets systemic, lymphoid, as well as mucosal tissues of rhesus macaques. HTLV-2 infection induces humoral as well as cell-mediated immune responses, and importantly, T-cell responses can be found at both systemic and mucosal sites. In this study, we demonstrate that the viral and T-cell dynamics of macaques dually infected with HTLV-2 and SIV_mac251 are similar to those of macaques singly infected with SIV_mac251.