Process of Infection with Bacteriophage φX174: XXXVI. Measurement of Virus-Specific Proteins During a Normal Cycle of Infection

Double-labeling techniques in which (14)C-labeled, phiX174-infected cells and (3)H-labeled, uninfected cells were used permitted the identification of the virus-specific proteins after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis without prior inhibition of host-cell protein synthesis by ultraviolet irradiation. It was also possible to detect previously undescribed components of high molecular weight which may represent induced host proteins. The gel regions specifically corresponding to cistron II protein and the chloramphenicol-resistant VI protein were identified, and a third new, small peak of unknown origin was detected. Studies of the rate of synthesis of virus-specific proteins at various times after infection indicated that the product of cistron I (lysis) is made only late in infection, but the other proteins seemed to be synthesized at the same relative rates throughout infection (although in different amounts). Studies of the proteins obtained from uniformly labeled phiX virus particles indicated that all of the spikes are identical and allowed a formulation of the structure of the phage capsid.     

Process of Infection with Bacteriophage φX174: XXXIV. Kinetics of the Attachment and Eclipse Steps of the Infection

The products of phiX cistrons II, III, and VII are demonstrated to affect the attachment of the phage to its host Escherichia coli C; therefore, by inference, these cistrons influence, directly or indirectly, the structure of proteins in the virus particle. Two of the mutations which alter attachment kinetics, ts79 in cistron III and h in cistron VII, also affect the electrophoretic mobility of the virus and emphasize the role of charge in the attachment interaction with the host. The kinetics for attached phage to go into "eclipse" are first-order and biphasic; about 85% of the phage eclipse at one rate (k(e) = 0.86 min(-1)) and the remainder do so at a distinctly lower rate (k(e) = 0.21 min(-1)). No phiX cistrons yet identified affect the eclipse process. The lowest temperature at which eclipse is detected is 19 C. The Arrhenius activation energy for phage eclipse has the high value of 36.6 kcal/mole, indicating the cooperative nature of the event.     

Measurement of γHV68 Infection in Mice

γ-Herpesviruses (γ-HVs) are notable for their ability to establish latent infections of lymphoid cells¹. The narrow host range of human γ-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine γ-herpesvirus 68 (γHV68) shares extensive genetic and biological similarities with human γ-HVs and is a natural pathogen of murid rodents². As such, evaluation of γHV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during γ-HVs infection.Upon intranasal inoculation, γHV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host^3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of γHV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.Download video file.^{(81M, mov)}     

Control of Murine Cytomegalovirus Infection by γδ T Cells

Infections with cytomegalovirus (CMV) can cause severe disease in immunosuppressed patients and infected newborns. Innate as well as cellular and humoral adaptive immune effector functions contribute to the control of CMV in immunocompetent individuals. None of the innate or adaptive immune functions are essential for virus control, however. Expansion of γδ T cells has been observed during human CMV (HCMV) infection in the fetus and in transplant patients with HCMV reactivation but the protective function of γδ T cells under these conditions remains unclear. Here we show for murine CMV (MCMV) infections that mice that lack CD8 and CD4 αβ-T cells as well as B lymphocytes can control a MCMV infection that is lethal in RAG-1^-/- mice lacking any T- and B-cells. γδ T cells, isolated from infected mice can kill MCMV infected target cells in vitro and, importantly, provide long-term protection in infected RAG-1^-/- mice after adoptive transfer. γδ T cells in MCMV infected hosts undergo a prominent and long-lasting phenotypic change most compatible with the view that the majority of the γδ T cell population persists in an effector/memory state even after resolution of the acute phase of the infection. A clonotypically focused Vγ1 and Vγ2 repertoire was observed at later stages of the infection in the organs where MCMV persists. These findings add γδ T cells as yet another protective component to the anti-CMV immune response. Our data provide clear evidence that γδ T cells can provide an effective control mechanism of acute CMV infections, particularly when conventional adaptive immune mechanisms are insufficient or absent, like in transplant patient or in the developing immune system in utero. The findings have implications in the stem cell transplant setting, as antigen recognition by γδ T cells is not MHC-restricted and dual reactivity against CMV and tumors has been described.     

What Triggers a Diagnosis of HIV Infection in the Tokyo Metropolitan Area? Implications for Preventing the Spread of HIV Infection in Japan

Background

Japan has not succeeded in reducing the annual number of new HIV-infected patients, although the prevalence of HIV infection is low (0.02%).

Methods

A single-center observational study was conducted at the largest HIV clinic in Tokyo, which treats 15% of the total patients in Japan, to determine the reasons for having diagnostic tests in newly infected individuals. HIV-infected patients who visited our clinic for the first time between 2011 and 2014 were analyzed.

Results

The 598 study patients comprised one-third of the total reported number of new patients in Tokyo during the study period. 76% were Japanese MSM. The reasons for being tested which led to the diagnosis was voluntary testing in 32%, existing diseases in 53% (AIDS-defining diseases in 22%, sexually transmitted infections (STI) in 8%, diseases other than AIDS or STIs in 23%) and routine pre-surgery or on admission screening in 15%. 52% and 74% of the study patients and patients presented with AIDS, respectively, had never been tested. The median CD4 count in patients with history of previous testing (315/μL) was significantly higher than that of patients who had never been tested (203/μL, p<0.001).

Conclusions

Only 32% of the newly HIV diagnosed patients were diagnosed because of voluntary testing, and 53% were diagnosed due to presence of other diseases. These results remain unchanged from our previous report 10 years earlier (2000–2004) on newly diagnosed patients at the same clinic. HIV testing has not been widely used by newly diagnosed patients in the Tokyo metropolitan area.     

Effects of Furazolidone on the Infection of Vibrio cholerae by Bacteriophage φ149

Furazolidone in concentrations which had little effect on the growth of host organisms greatly reduced the yield of phage 149 from the host Vibrio cholerae OGAWA 154. This phage was resistant to the in vitro action of the drug. The phage yield of infected bacteria depended significantly on the time of addition or withdrawal of the drug. The average burst size of the drug-treated and infected bacteria decreased exponentially with increase in drug concentration. The latent period of phage multiplication and also the eclipse period did not change significantly from the control values. A concentration of 0.05 μg of furazolidone per ml inhibited DNA synthesis by about 50% in phage-infected cells and only by about 18% in noninfected ones, relative to the respective controls. RNA and protein synthesis were affected by a much smaller degree both in infected and noninfected cells. Quantitative deduction of the length of furazolidone-treated cells from their phage adsorption characteristics and its agreement with previous electron microscopy data indicated that furazolidone did not affect the phage receptors.     

Etiology of Sarcoidosis: Does Infection Play a Role?

Sarcoidosis is a granulomatous inflammatory disorder of unclear etiology, which is known to affect multiple organ systems including the lungs, heart, skin, central nervous system, and eyes, among others. For this reason, sarcoidosis represents a systemic medical disorder that is clinically relevant to multiple medical sub-specialties. Despite extensive research, the etiology of sarcoidosis has yet to be elucidated, although most evidence supports that the pathogenetic mechanism of sarcoidosis is an aberrant immune response, driven by an unidentified antigen (or antigens) in genetically susceptible individuals. Multiple candidate etiologic agents, including microbial organisms and environmental agents, have been investigated, but study results are inconclusive. In this review, we describe the known histologic and immunologic features of sarcoidosis and discuss the evidence supporting a role for infectious processes in the pathogenesis of sarcoidosis.     

Viral Bcl-2-Mediated Evasion of Autophagy Aids Chronic Infection of γHerpesvirus 68

γ-herpesviruses (γHVs) have developed an interaction with their hosts wherein they establish a life-long persistent infection and are associated with the onset of various malignancies. One critical virulence factor involved in the persistency of murine γ-herpesvirus 68 (γHV68) is the viral homolog of the Bcl-2 protein (vBcl-2), which has been implicated to counteract both host apoptotic responses and autophagy pathway. However, the relative significance of the two activities of vBcl-2 in viral persistent infection has yet to be elucidated. Here, by characterizing a series of loss-of-function mutants of vBcl-2, we have distinguished the vBcl-2-mediated antagonism of autophagy from the vBcl-2-mediated inhibition of apoptosis in vitro and in vivo. A mutant γHV68 virus lacking the anti-autophagic activity of vBcl-2 demonstrates an impaired ability to maintain chronic infections in mice, whereas a mutant virus lacking the anti-apoptotic activity of vBcl-2 establishes chronic infections as efficiently as the wild-type virus but displays a compromised ability for ex vivo reactivation. Thus, the vBcl-2-mediated antagonism of host autophagy constitutes a novel mechanism by which γHVs confer persistent infections, further underscoring the importance of autophagy as a critical host determinant in the in vivo latency of γ-herpesviruses.     

Subterranean Mammals: Reservoirs of Infection or Overlooked Sentinels of Anthropogenic Environmental Soiling?

Global reports of emergent pathogens in humans have intensified efforts to identify wildlife reservoirs. Subterranean mammals, such as bathyergid mole rats, are largely overlooked, despite their high-level exposure to soil-dwelling microbes. Initial assessment of bathyergid reservoir potential was determined using a broad-range 16S rRNA PCR approach, which revealed an 83% PCR-positivity for the 234 bathyergid lung samples evaluated. The presence of the Bacillus cereus complex, a ubiquitous bacterial assemblage, containing pathogenic and zoonotic species, was confirmed through nucleotide sequencing, prior to group- and species-specific PCR sequencing. The latter allowed for enhanced placement and prevalence estimations of Bacillus in four bathyergid species sampled across a range of transformed landscapes in the Western Cape Province, South Africa. Two novel Bacillus strains (1 and 2) identified on the basis of the concatenated 16S rRNA-groEL-yeaC data set (2066 nucleotides in length), clustered with B. mycoides (ATCC 6462) and B. weihenstephanensis (WSBC 10204), within a well-supported monophyletic lineage. The levels of co-infection, evaluated with a groEL strain-specific assay, developed specifically for this purpose, were high (71%). The overall Bacillus presence of 17.95% (ranging from 0% for Georychus capensis to 45.35% for Bathyergus suillus) differed significantly between host species (χ² = 69.643; df = 3; P < 0.05), being significantly higher in bathyergids sampled near an urban informal settlement (χ² = 70.245; df = 3; P < 0.05). The results highlight the sentinel potential of soil-dwelling mammals for monitoring anthropogenically introduced, opportunistic pathogens and the threats they pose to vulnerable communities, particularly in the developing world.     

Abortive Infection of Sporulating Bacillus subtilis 168 by φ2 Bacteriophage

Bacteriophage phi2 is unable to replicate in Bacillus subtilis 168. Although some phage deoxyribonucleic acid (DNA) synthesis can occur, the DNA made is not biologically active and sedimentation analysis reveals that it is smaller in size than that of mature DNA or DNA isolated from phi2-infected permissive hosts. Messenger ribonucleic acid hybridizable with phi2 DNA is also synthesized in phi2-infected cells of 168. Mutants of 168 which are permissive hosts for phi2 have been isolated. These mutants are defective in sporulation and possess the phenotype of "early sporulation mutants." The majority map in two locations, one near the lys locus opposite the trp locus (spoA locus) and the other tightly linked to a phe locus.     

Hepatitis B Virus Infection—Current Concepts of Chronicity and Immunity

Among the three types of viral hepatitis agents—A, B and non-A, non-B—the hepatitis B virus (HBV) has been best characterized by immunologic and recombinant DNA technologies. The indefinite persistence of hepatitis B virus infection in 85% to 90% of perinatally infected infants and in about 10% of those infected later in life accounts for a worldwide epidemiologic reservoir of more than 200 million carriers who are at a high risk for the development of δ-infection, chronic liver disease and hepatocellular carcinoma. Active immunization with a safe and effective vaccine, derived from the plasma of carriers of hepatitis B surface antigen (HBsAg), is envisaged to avoid viral hepatitis type B and its chronic sequelae. In addition to serologic and immunohistochemical markers of hepatitis B virus infection, hybridization assays using cloned HBV DNA have provided new insight into the biology of this virus, its persistence and its oncogenic potential in humans and in animal models. Genetic similarities have been recognized between HBV and the antigenically distinct non-A, non-B agents implicated in some cases of transfusion-associated chronic hepatitis. Structurally this unique group of HBV and HBV-like agents are DNA viruses with functional attributes of integration and replication analogous to the retroviruses.     

Field Infection of Zonocerus variegatus following application of an oil‐based formulation of Metarhizium flavoviride conidia

Conidia of Metarhizium flavoviride grown in diphasic culture with sporulation on rice, were extracted in kerosene and formulated with peanut oil. The formulation led to high mortality when applied to field populations of Zonocerus variegatus; between 70 and 95% of insects in samples collected from the field 0, 3 or 7 days after spraying died. Sporulation of M. flavoviride was observed in 70–80% of the dead‐treated insects, which compares favourably with results obtained in laboratory bioassays. Many dead Z. variegatus, some with internal sporulation, were found in shaded niches and oviposition sites in the treated plots. No sporulation was observed in samples collected prior to application, and both sporulation and mortality differed significantly between control and treated plots. Some infections were observed in insects collected 16 and 23 days after treatment; these late infections may be a consequence of contact with spores surviving in the environment.     

Role for β2-Microglobulin in Echovirus Infection of Rhabdomyosarcoma Cells

A monoclonal antibody (MAb) that blocks most echoviruses (EVs) from infecting rhabdomyosarcoma (RD) cells has been isolated. By using the CELICS cloning method (T. Ward, P. A. Pipkin, N. A. Clarkson, D. M. Stone, P. D. Minor, and J. W. Almond, EMBO J. 13:5070–5074, 1994), the ligand for this antibody has been identified as β2-microglobulin (β2m), the 12-kDa protein that associates with class I heavy chains to form class I HLA complexes. A commercial MAb (MAb 1350) against β2m was also found to block EV7 infection without affecting binding to its receptor, DAF, or replication of EV7 viral RNA inside cells. Entry of EV7 into cells was reduced by only 30% by antibody and cytochalasin D, an inhibitor of endocytosis mediated by caveolae and clathrin-coated pits, but was not significantly reduced by sodium azide. The block to virus entry by cytochalasin D was additive to the block induced by antibody. We suggest that EV7 rapidly enters into a multicomponent receptor complex prior to entry into cells and that this initial entry event requires β2m or class I HLA for infection to proceed.Echoviruses (EVs) are members of the Enterovirus genus of the family Picornaviridae and are important human pathogens. They are associated with a wide spectrum of clinical syndromes, including rashes, diarrhea, aseptic meningitis, respiratory disease, and possibly conditions such as chronic fatigue syndrome. This range of clinical manifestations is probably a reflection of virus tissue tropisms, which seem to be mediated, at least in part, by utilization of a range of cellular receptors.Anti-cell surface monoclonal antibodies (MAbs) that block EV infection have been isolated previously and have been used to determine the identity of some of these receptors. In 1992 Bergelson et al. demonstrated that EV serotypes 1 and 8 use the collagen receptor VLA-2 (6) by attaching to the α2 subunit (7). Previously, we and others have shown that a regulator of complement activity, decay-accelerating factor (DAF), is the receptor for a range of hemagglutinating EVs (3, 37). Other EVs appear to use neither of these, but the identity of their receptor(s) is unknown. Mbida et al. have isolated a MAb (MAb 143) that blocks most EV serotypes from infecting a range of cell types. MAb 143 was also found to block coxsackievirus A9 but not poliovirus or coxsackievirus serotypes B1 to B6 (21). The ligand for MAb 143 was found by affinity purification to be an unknown 44-kDa glycoprotein (22). It was therefore suggested that the 44-kDa protein was part of a multicomponent receptor complex used by most EVs to infect cells. A direct role for the 44-kDa protein in virus attachment seems unlikely, since MAb 143 blocks infection by the viruses that have been shown to use other proteins, such as DAF (3, 37) and VLA-2 (6), as their primary receptors.Here, we report the isolation of a MAb similar to that described by Mbida et al. (21, 22) and describe the cloning and identification of its ligand. The ligand is β2-microglobulin (β2m), a 12-kDa protein that associates with the class I HLA heavy chains (44 kDa) and presents antigenic peptides (20). We show that MAbs to β2m block EV infection partly by reducing the entry of virus into cells, although other postbinding effects cannot be ruled out.     

The Biology of Kaposi＇s Sarcoma-Associated Herpesvirus and the Infection of Human Immunodeficiency Virus

Kaposi sarcoma-associated herpesvirus (KSHV),also known as human herpesvirus 8 (HHV-8),is discovered in 1994 from Kaposi's sarcoma (KS) lesion of an acquired immunodeficiency syndrome (AIDS)patient.In addition to its association with KS,KSHV has also been implicated as the causative agent of two other AIDS-associated malignancies:primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD).KSHV is a complex DNA virus that not only has the ability to promote cellular growth and survival for tumor development,but also can provoke deregulated angiogenesis,inflammation,and modulate the patient's immune system in favor of tumor growth.As KSHV is a necessary but not sufficient etiological factor for KS,human immunodeficiency virus (HIV) is a very important cofactor.Here we review the basic information about the biology of KSHV,development of pathogenesis and interaction between KSHV and HIV.     

Does Rust Infection of Willow Affect Feeding and Oviposition Behavior of Willow Leaf Beetles?

Willows are often attacked by both herbivorous insects and rust fungi. Little is known about interactions between these two willow enemies. We studied whether feeding and oviposition behavior of the willow leaf beetle Plagiodera versicolora upon the willow hybrid Salix x cuspidata is affected when the rust fungus Melampsora allii-fragilis has attacked the plant. Laboratory bioassays revealed that adult willow leaf beetles significantly avoided feeding and oviposition on rust-infected leaves when compared to healthy leaves. Further bioassays aimed to elucidate the temporal and spatial scale of effects of rust infection on feeding behavior of adults. While infected parts of leaves were avoided at all times past infection tested (8, 12, and 16 days), symptom-free parts of infected leaves were only avoided 16 days past infection. Systemic effects extended only one leaf position up and two leaf positions down from the infection site.     

Bacteriophage Φ S1 Infection of Pseudomonas fluorescens Planktonic Cells versus Biofilms

This communication focuses on the efficacy of a specific lytic phage, phage F S1, as a control agent of Pseudomonas fluorescens biofilms. The effect of phage infection temperature and the host growth temperature were evaluated. The results obtained showed that the phage infection process was temperature dependent and that the optimum temperature of infection of planktonic cells and biofilms was 26°C. At this temperature, bacteriophage F S1, at a multiplicity of infection (MOI) of 0.5 infected both planktonic cells and biofilms causing a biomass reduction of about 85% in both cases.     

Mechanisms of the Blood–Brain Barrier Disruption in HIV-1 Infection

Summary 1. Alterations of brain microvasculature and the disruption of the blood–brain barrier (BBB) integrity are commonly associated with human immunodeficiency virus type 1 (HIV-1) infection. These changes are most frequently found in human immunodeficiency virus-related encephalitis (HIVE) and in human immunodeficiency virus-associated dementia (HAD).2. It has been hypothesized that the disruption of the BBB occurs early in the course of HIV-1 infection and can be responsible for HIV-1 entry into the CNS.3. The current review discusses the mechanisms of injury to brain endothelial cells and alterations of the BBB integrity in HIV-infection with focus on the vascular effects of HIV Tat protein. In addition, this review describes the mechanisms of the BBB disruption due to HIV-1 or Tat protein interaction with selected risk factors for HIV infection, such as substance abuse and aging.This revised article was published online in May 2005 with a February 2005 cover date.