Mapping the immune response to the outer domain of a human immunodeficiency virus-1 clade C gp120

The outer domain (OD) of human immunodeficiency virus (HIV)-1 gp120 represents an attractive, if difficult, target for a beneficial immune response to HIV infection. Unlike the entire gp120, the OD is structurally stable and contains the surfaces that interact with both the primary and secondary cellular receptors. The primary strain-specific neutralizing target, the V3 loop, lies within the OD, as do epitopes for two cross-reactive neutralizing monoclonal antibodies (mAbs), b12 and 2G12, and the contact sites for a number of inhibitory lectins. The OD is poorly immunogenic, at least in the context of complete gp120, but purposeful OD immunization can lead to a substantial antibody response. Here, we map the antibody generated following immunization with a clade C OD. In contrast to published data for the clade B OD, the majority of the polyclonal response to the complete clade C OD is to the V3 loop; deletion of the loop substantially reduces immunogenicity. When the loop sequence was substituted for the epitope for 2F5, a well-characterized human cross-neutralizing mAb, a polyclonal response to the epitope was generated. A panel of mAbs against the clade C OD identified two mAbs that reacted with the loop and were neutralizing for clade C but not B isolates. Other mAbs recognized both linear and conformational epitopes in the OD. We conclude that, as for complete gp120, V3 immunodominance is a property of OD immunogens, that the responses can be neutralizing and that it could be exploited for the presentation of other epitopes.

Broadly neutralizing antibodies protect against hepatitis C virus quasispecies challenge

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Antiviral effects of antisense morpholino oligomers in murine coronavirus infection models

The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell culture, and one of these (5TERM), which was complementary to the 5' terminus of the genomic RNA, was effective against six strains of MHV. Further studies were carried out with various arginine-rich peptides conjugated to the 5TERM PMO sequence in order to evaluate efficacy and toxicity and thereby select candidates for in vivo testing. In uninfected mice, prolonged P-PMO treatment did not result in weight loss or detectable histopathologic changes. 5TERM P-PMO treatment reduced viral titers in target organs and protected mice against virus-induced tissue damage. Prophylactic 5TERM P-PMO treatment decreased the amount of weight loss associated with infection under most experimental conditions. Treatment also prolonged survival in two lethal challenge models. In some cases of high-dose viral inoculation followed by delayed treatment, 5TERM P-PMO treatment was not protective and increased morbidity in the treated group, suggesting that P-PMO may cause toxic effects in diseased mice that were not apparent in the uninfected animals. However, the strong antiviral effect observed suggests that with further development, P-PMO may provide an effective therapeutic approach against a broad range of coronavirus infections.

Reduced incorporation of the influenza B virus BM2 protein in virus particles decreases infectivity

BM2 is the fourth integral membrane protein encoded by the influenza B virus genome. It is synthesized late in infection and transported to the plasma membrane from where it is subsequently incorporated into progeny virus particles. It has recently been reported that BM2 has ion channel activity and may be the functional homologue of the influenza A virus M2 protein acting as an ion channel involved in viral entry. Using a reverse genetic approach it was not possible to recover virus which lacked BM2. A recombinant influenza B virus was generated in which the BM2 AUG initiation codon was mutated to GUG. This decreased the efficiency of translation of BM2 protein such that progeny virions contained only 1/8 the amount of BM2 seen in wild-type virus. The reduction in BM2 incorporation resulted in a reduction in infectivity although there was no concomitant decrease in the numbers of virions released from the infected cells. These data imply that the incorporation of sufficient BM2 protein into influenza B virions is required for infectivity of the virus particles. (C) 2004 Elsevier Inc. All rights reserved.

Interactions of decay-accelerating factor (DAF) with haemagglutinating human enteroviruses: utilizing variation in primate DAF to map virus binding sites

A cellular receptor for the haemagglutinating enteroviruses (HEV), and the protein that mediates haemagglutination, is the membrane complement regulatory protein decay accelerating factor (DAF; CD55). Although primate DAF is highly conserved, significant differences exist to enable cell lines derived from primates to be utilized for the characterization of the DAF binding phenotype of human enteroviruses. Thus, several distinct DAF-binding phenotypes of a selection of HEVs (viz. coxsackievirus A21 and echoviruses 6, 7, 11-13, 29) were identified from binding and infection assays using a panel of primate cells derived from human, orang-utan, African Green monkey and baboon tissues. These studies complement our recent determination of the crystal structure of SCR(34) of human DAF [Williams, P., Chaudhry, Y., Goodfellow, I. G., Billington, J., Powell, R., Spiller, O. B., Evans, D. J. & Lea, S. (2003). J Biol Chem 278, 10691-10696] and have enabled us to better map the regions of DAF with which enteroviruses interact and, in certain cases, predict specific virus-receptor contacts.

Non-promoting diterpene esters can induce Epstein-Barr Virus early antigen expression in the Raji cell line

A range of diterpene ester ligands with selective biological activity (e.g., irritant but not tumour promoting) were tested for their ability to induce Epstein-Barr virus (EBV) early antigen expression in the lymphoblastoid Raji cell line. All substituted compounds were found to be capable of inducing some antigen expression at nM−μM levels, including desacetyl-α-sapinine, a compound largely devoid of biological activity. The non-promoting, fluorescent compound, sapintoxin A, was virtually equipotent with promoting compounds. It was concluded that, although the assay has relevance to the specific condition of chronic diterpene ester exposure occurring in conjunction with high EBV infection rates, there was relatively poor correlation with mouse skin tumour promoting potential.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Human enhancement: could you become infected with a computer virus?

Experiments demonstrating human enhancement through the implantation of technology in healthy humans have been performed for over a decade by some academic research groups. More recently, technology enthusiasts have begun to realize the potential of implantable technology such as glass capsule RFID transponders. In this paper it is argued that implantable RFID devices have evolved to the point whereby we should consider the devices themselves as simple computers. Presented here is the infection with a computer virus of an RFID device implanted in a human. Coupled with our developing concept of what constitutes the human body and its boundaries, it is argued that this study has given rise to the world’s first human infected with a computer virus. It has taken the wider academic community some time to agree that meaningful discourse on the topic of implantable technology is of value. As developments in medical technologies point to greater possibilities for enhancement, this shift in thinking is not too soon in coming.

Enhanced hepatitis C virus genome replication and lipid accumulation mediated by inhibition of AMP-activated protein kinase

Hepatitis C virus (HCV) infection is associated with dysregulation of both lipid and glucose metabolism. As well as contributing to viral replication, these perturbations influence the pathogenesis associated with the virus, including steatosis, insulin resistance, and type 2 diabetes. AMP-activated protein kinase (AMPK) plays a key role in regulation of both lipid and glucose metabolism. We show here that, in cells either infected with HCV or harboring an HCV subgenomic replicon, phosphorylation of AMPK at threonine 172 and concomitant AMPK activity are dramatically reduced. We demonstrate that this effect is mediated by activation of the serine/threonine kinase, protein kinase B, which inhibits AMPK by phosphorylating serine 485. The physiological significance of this inhibition is demonstrated by the observation that pharmacological restoration of AMPK activity not only abrogates the lipid accumulation observed in virus-infected and subgenomic replicon-harboring cells but also efficiently inhibits viral replication. These data demonstrate that inhibition of AMPK is required for HCV replication and that the restoration of AMPK activity may present a target for much needed anti-HCV therapies.

Suppression of a pro-apoptotic K+ channel as a mechanism for hepatitis C virus persistence

An estimated 3% of the global population are infected with hepatitis C virus (HCV), and the majority of these individuals will develop chronic liver disease. As with other chronic viruses, establishment of persistent infection requires that HCV-infected cells must be refractory to a range of pro-apoptotic stimuli. In response to oxidative stress, amplification of an outward K(+) current mediated by the Kv2.1 channel, precedes the onset of apoptosis. We show here that in human hepatoma cells either infected with HCV or harboring an HCV subgenomic replicon, oxidative stress failed to initiate apoptosis via Kv2.1. The HCV NS5A protein mediated this effect by inhibiting oxidative stress-induced p38 MAPK phosphorylation of Kv2.1. The inhibition of a host cell K(+) channel by a viral protein is a hitherto undescribed viral anti-apoptotic mechanism and represents a potential target for antiviral therapy.

Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis

Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K+ channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A in order to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K+ homeostasis.

Modulation of potassium channels inhibits bunyavirus infection

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K+) channels to infect cells. Time of addition assays using K+ channel modulating agents demonstrated that K+ channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K+ channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, twin-pore domain K+ channels (K2P) were identified as the K+ channel family mediating BUNV K+ channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Epstein-Barr virus: clinical and epidemiological revisits and genetic basis of oncogenesis

Epstein-Barr virus (EBV) is classified as a member in the order herpesvirales, family herpesviridae, subfamily gammaherpesvirinae and the genus lymphocytovirus. The virus is an exclusively human pathogen and thus also termed as human herpesvirus 4 (HHV4). It was the first oncogenic virus recognized and has been incriminated in the causation of tumors of both lymphatic and epithelial nature. It was reported in some previous studies that 95% of the population worldwide are serologically positive to the virus. Clinically, EBV primary infection is almost silent, persisting as a life-long asymptomatic latent infection in B cells although it may be responsible for a transient clinical syndrome called infectious mononucleosis. Following reactivation of the virus from latency due to immunocompromised status, EBV was found to be associated with several tumors. EBV linked to oncogenesis as detected in lymphoid tumors such as Burkitt's lymphoma (BL), Hodgkin's disease (HD), post-transplant lymphoproliferative disorders (PTLD) and T-cell lymphomas (e.g. Peripheral T-cell lymphomas; PTCL and Anaplastic large cell lymphomas; ALCL). It is also linked to epithelial tumors such as nasopharyngeal carcinoma (NPC), gastric carcinomas and oral hairy leukoplakia (OHL). In vitro, EBV many studies have demonstrated its ability to transform B cells into lymphoblastoid cell lines (LCLs). Despite these malignancies showing different clinical and epidemiological patterns when studied, genetic studies have suggested that these EBV- associated transformations were characterized generally by low level of virus gene expression with only the latent virus proteins (LVPs) upregulated in both tumors and LCLs. In this review, we summarize some clinical and epidemiological features of EBV- associated tumors. We also discuss how EBV latent genes may lead to oncogenesis in the different clinical malignancies

Sole infection by human metapneumovirus among children with radiographically diagnosed community-acquired pneumonia in a tropical region

Background Limited information is available on the role of human metapneumovirus (HMPV) as the unique pathogen among children hospitalized for community-acquired pneumonia (CAP) in a tropical region. Objective We aimed to describe HMPV infection among children with CAP investigating bacterial and viral co-infections. Patients and methods A prospective study was carried out in Salvador, North-East Brazil. Overall, 268 children aged <5 years hospitalized for CAP were enrolled. Human metapneumovirus RNA was detected in nasopharyngeal aspirates (NPA) by reverse transcription polymerase chain reaction. Sixteen other bacterial and viral pathogens were investigated by an expanded panel of laboratory methods. Chest X-ray taken on admission was read by an independent paediatric radiologist unaware of clinical information or the established aetiology. Results Human metapneumovirus RNA was detected in NPAs of 11 (4.1%) children, of which 4 (36%) had sole HMPV infection. The disease was significantly shorter among patients with sole HMPV infection in comparison with patients with mixed infection (4 +/- 1 versus 7 +/- 2 days, P = 0.03). Three of those four patients had alveolar infiltrates. Conclusion Sole HMPV infection was detected in children with CAP in Salvador, North-East Brazil. HMPV may play a role in the childhood CAP burden.

Multilevel analysis of hepatitis A infection in children and adolescents: a household survey in the Northeast and Central-west regions of Brazil

Background The objectives were to estimate the prevalence of hepatitis A among children and adolescents from the Northeast and Midwest regions and the Federal District of Brazil and to identify individual-, household- and area-levels factors associated with hepatitis A infection. Methods This population-based survey was conducted in 20042005 and covered individuals aged between 5 and 19 years. A stratified multistage cluster sampling technique with probability proportional to size was used to select 1937 individuals aged between 5 and 19 years living in the Federal capital and in the State capitals of 12 states in the study regions. The sample was stratified according to age (59 and 10- to 19-years-old) and capital within each region. Individual- and household-level data were collected by interview at the home of the individual. Variables related to the area were retrieved from census tract data. The outcome was total antibodies to hepatitis A virus detected using commercial EIA. The age distribution of the susceptible population was estimated using a simple catalytic model. The associations between HAV infection and independent variables were assessed using the odds ratio and corrected for the random design effect and sampling weight. Multilevel analysis was performed by GLLAMM using Stata 9.2. Results The prevalence of hepatitis A infection in the 59 and 1019 age-group was 41.5 and 57.4, respectively for the Northeast, 32.3 and 56.0, respectively for the Midwest and 33.8 and 65.1 for the Federal District. A trend for the prevalence of HAV infection to increase according to age was detected in all sites. By the age of 5, 31.5 of the children had already been infected with HAV in the Northeast region compared with 20.0 in the other sites. By the age of 19 years, seropositivity was 70 in all areas. The curves of susceptible populations differed from one area to another. Multilevel modeling showed that variables relating to different levels of education were associated with HAV infection in all sites. Conclusion The study sites were classified as areas with intermediate endemicity area for hepatitis A infection. Differences in age trends of infection were detected among settings. This multilevel model allowed for quantification of contextual predictors of hepatitis A infection in urban areas.