Site 1 protease is required for proteolytic processing of the glycoproteins of the South American hemorrhagic fever viruses Junin, Machupo, and Guanarito

The cellular proprotein convertase site 1 protease (S1P) has been implicated in the proteolytic processing of the glycoproteins (GPs) of Old World arenaviruses. Here we report that S1P is also involved in the processing of the GPs of the genetically more-distant South American hemorrhagic fever viruses Guanarito, Machupo, and Junin. Efficient cleavage of Guanarito virus GP, whose protease recognition sites deviate from the reported S1P consensus sequence, indicates a broader specificity of S1P than anticipated. Lack of GP processing of Junin virus dramatically reduced production of infectious virus and prevented cell-to-cell propagation. Infection of S1P-deficient cells resulted in viral persistence over several weeks without the emergence of escape variants able to use other cellular proteases for GP processing.     

Cell entry by human pathogenic arenaviruses

The arenaviruses Lassa virus (LASV) in Africa and Machupo (MACV), Guanarito (GTOV) and Junin viruses (JUNV) in South America cause severe haemorrhagic fevers in humans with fatality rates of 15–35%. The present review focuses on the first steps of infection with human pathogenic arenaviruses, the interaction with their cellular receptor molecules and subsequent entry into the host cell. While similarities exist in genomic organization, structure and clinical disease caused by pathogenic Old World and New World arenaviruses these pathogens use different primary receptors. The Old World arenaviruses employ α-dystroglycan, a cellular receptor for proteins of the extracellular matrix, and the human pathogenic New World arenaviruses use the cellular cargo receptor transferrin receptor 1. While the New World arenavirus JUNV enters cells via clathrin-dependent endocytosis, evidence occurred for clathrin-independent entry of the prototypic Old World arenavirus lymphocytic choriomeningitis virus. Upon internalization, arenaviruses are delivered to the endosome, where pH-dependent membrane fusion is mediated by the envelope glycoprotein (GP). While arenavirus GPs share characteristics with class I fusion GPs of other enveloped viruses, unusual mechanistic features of GP-mediated membrane fusion have recently been discovered for arenaviruses with important implications for viral entry.     

Arenoviruses in Vero Cells: Ultrastructural Studies

Thin-section electron microscopy was carried out on Vero green monkey kidney cell cultures infected with some viruses of the newly constituted arenovirus group. Junin, Machupo, Amapari, Pichinde, Parana, Tamiami, and Latino viruses were morphologically identical and indistinguishable from lymphocytic choriomeningitis virus, the prototype virus of the group. Virus particles were round, oval, or pleomorphic, 60 to 280 nm in diameter, and matured via budding from plasma membranes. Most characteristically, particles contained various amounts of homogeneous, 20- to 25-nm, dense granules; these granules in large masses also formed distinctive intracytoplasmic inclusions. In negative-contrast preparations from infected Vero cell culture supernatant fluids, several of the viruses appeared as pleomorphic membrane-bound forms with rather pronounced surface projections. Most particles were between 90 and 220 nm in diameter, although some reached 350 nm in their longest dimension. Internal structure was not resolved by negative-contrast electron microscopy. All observations supported the current delineation of a distinct arenovirus group.     

Unique small molecule entry inhibitors of hemorrhagic fever arenaviruses

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.     

A Multivalent and Cross-Protective Vaccine Strategy against Arenaviruses Associated with Human Disease

Arenaviruses are the causative pathogens of severe hemorrhagic fever and aseptic meningitis in humans, for which no licensed vaccines are currently available. Pathogen heterogeneity within the Arenaviridae family poses a significant challenge for vaccine development. The main hypothesis we tested in the present study was whether it is possible to design a universal vaccine strategy capable of inducing simultaneous HLA-restricted CD8⁺ T cell responses against 7 pathogenic arenaviruses (including the lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses), either through the identification of widely conserved epitopes, or by the identification of a collection of epitopes derived from multiple arenavirus species. By inoculating HLA transgenic mice with a panel of recombinant vaccinia viruses (rVACVs) expressing the different arenavirus proteins, we identified 10 HLA-A02 and 10 HLA-A03-restricted epitopes that are naturally processed in human antigen-presenting cells. For some of these epitopes we were able to demonstrate cross-reactive CD8⁺ T cell responses, further increasing the coverage afforded by the epitope set against each different arenavirus species. Importantly, we showed that immunization of HLA transgenic mice with an epitope cocktail generated simultaneous CD8⁺ T cell responses against all 7 arenaviruses, and protected mice against challenge with rVACVs expressing either Old or New World arenavirus glycoproteins. In conclusion, the set of identified epitopes allows broad, non-ethnically biased coverage of all 7 viral species targeted by our studies.     

New World arenavirus clade C, but not clade A and B viruses, utilizes alpha-dystroglycan as its major receptor

Alpha-dystroglycan (alpha-DG) has been identified as a major receptor for lymphocytic choriomeningitis virus (LCMV) and Lassa virus, two Old World arenaviruses. The situation with New World arenaviruses is less clear: previous studies demonstrated that Oliveros virus also exhibited high-affinity binding to alpha-DG but that Guanarito virus did not. To extend these initial studies, several additional Old and New World arenaviruses were screened for entry into mouse embryonic stem cells possessing or lacking alpha-DG. In addition, representative viruses were further analyzed for direct binding to alpha-DG by means of a virus overlay protein blot assay technique. These studies indicate that Old World arenaviruses use alpha-DG as a major receptor, whereas, of the New World arenaviruses, only clade C viruses (i.e., Oliveros and Latino viruses) use alpha-DG as a major receptor. New World clade A and B arenaviruses, which include the highly pathogenic Machupo, Guanarito, Junin, and Sabia viruses, appear to use a different receptor or coreceptor for binding. Previous studies with LCMV have suggested the need for a small aliphatic amino acid at LCMV GP1 glycoprotein amino acid position 260 to allow high-affinity binding to alpha-DG. As reported herein, this requirement appears to be broadly applicable to the arenaviruses as determined by more extensive analysis of alpha-DG receptor usage and GP1 sequences of Old and New World arenaviruses. In addition, GP1 amino acid position 259 also appears to be important, since all arenaviruses showing high-affinity alpha-DG binding possess a bulky aromatic amino acid (tyrosine or phenylalanine) at this position.     

Common Antiviral Cytotoxic T-Lymphocyte Epitope for Diverse Arenaviruses

Members of the Arenaviridae family have been isolated from mammalian hosts in disparate geographic locations, leading to their grouping as Old World types (i.e., lymphocytic choriomeningitis virus [LCMV], Lassa fever virus [LFV], Mopeia virus, and Mobala virus) and New World types (i.e., Junin, Machupo, Tacaribe, and Sabia viruses) (C. J. Peters, M. J. Buchmeier, P. E. Rollin, and T. G. Ksiazek, p. 1521-1551, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996; P. J. Southern, p. 1505-1519, in B. N. Fields, D. M. Knipe, and P. M. Howley [ed.], Fields virology, 3rd ed., 1996). Several types in both groups-LFV, Junin, Machupo, and Sabia viruses-cause severe and often lethal human diseases. By sequence comparison, we noted that eight Old World and New World arenaviruses share several amino acids with the nucleoprotein (NP) that consists of amino acids (aa) 118 to 126 (NP 118-126) (RPQASGVYM) of LCMV that comprise the immunodominant cytotoxic T-lymphocyte (CTL) epitope for H-2(d) mice (32). This L(d)-restricted epitope constituted >97% of the total bulk CTLs produced in the specific antiviral or clonal responses of H-2(d) BALB mice. NP 118-126 of the Old World arenaviruses LFV, Mopeia virus, and LCMV and the New World arenavirus Sabia virus bound at high affinity to L(d). The primary H-2(d) CTL anti-LCMV response as well as that of a CTL clone responsive to LCMV NP 118-126 recognized target cells coated with NP 118-126 peptides derived from LCMV, LFV, and Mopeia virus but not Sabia virus, indicating that a common functional NP epitope exists among Old World arenaviruses. Use of site-specific amino acid exchanges in the NP CTL epitope among these arenaviruses identified amino acids involved in major histocompatibility complex binding and CTL recognition.     

Template activity of poly(A+) and poly(A-) messenger RNA of pathogenic arenaviruses

The total RNA from cells infected with Machupo and Lassa viruses as well as poly(A+) and poly(A-) fractions of the RNA were translated in the cell-free protein synthesizing system from rabbit reticulocytes. The translated products were treated with specific antibodies and analyzed in polyacrylamide gel electrophoresis. Only poly(A-) fraction of RNA coded for the synthesis of NP protein in vitro. The mRNAs for NP protein of Machupo and Lassa viruses are supposed to contain no poly(A) sequences at 3'end, or if they really do, the size of the sequences is not adequate for binding with oligo(dT)-cellulose.     

Codon usage signatures in Sabia and Chapare for host adaptation

Sabia and Chapare viruses in the Arenavirus family cause viral hemorrhagic fever among humans with a fatality rate of 30% with no treatment models. Therefore, it is of interest to document the codon usage, amino acid patterns and associated factors influencing the observed variations in Sabia and Chapare viruses for host adaptation. Multivariate statistical analysis revealed compositional constraint and host selection pressure influencing the viral codon usage patterns. These data suggests the codon usage signatures in Sabia and Chapare viruses for host adaptation in the human host implying its role in the rapid progression of the infection. Dinucleotides UpG and CpA were noted to be over-represented among the Sabia, Chapare viruses and human genomes. Strong restraint from the usage of CpG dinucleotides among viruses is linked with the molecular mimicry of the human immune system. Thus, the data reported from this study help in understanding the mechanism of viral adaptation inside the host genome for further consideration in drug discovery.     

The emergence of pandemic influenza viruses

Pandemic influenza has posed an increasing threat to public health worldwide in the last decade. In the 20th century, three human pandemic influenza outbreaks occurred in 1918, 1957 and 1968, causing significant mortality. A number of hypotheses have been proposed for the emergence and development of pandemic viruses, including direct introduction into humans from an avian origin and reassortment between avian and previously circulating human viruses, either directly in humans or via an intermediate mammalian host. However, the evolutionary history of the pandemic viruses has been controversial, largely due to the lack of background genetic information and rigorous phylogenetic analyses. The pandemic that emerged in early April 2009 in North America provides a unique opportunity to investigate its emergence and development both in human and animal aspects. Recent genetic analyses of data accumulated through long-term influenza surveillance provided insights into the emergence of this novel pandemic virus. In this review, we summarise the recent literature that describes the evolutionary pathway of the pandemic viruses. We also discuss the implications of these findings on the early detection and control of future pandemics.     

Polyfunctional CD4+ T cell responses to a set of pathogenic arenaviruses provide broad population coverage

Background

Several arenaviruses cause severe hemorrhagic fever and aseptic meningitis in humans for which no licensed vaccines are available. A major obstacle for vaccine development is pathogen heterogeneity within the Arenaviridae family. Evidence in animal models and humans indicate that T cell and antibody-mediated immunity play important roles in controlling arenavirus infection and replication. Because CD4⁺ T cells are needed for optimal CD8⁺ T cell responses and to provide cognate help for B cells, knowledge of epitopes recognized by CD4⁺ T cells is critical to the development of an effective vaccine strategy against arenaviruses. Thus, the goal of the present study was to define and characterize CD4⁺ T cell responses from a broad repertoire of pathogenic arenaviruses (including lymphocytic choriomeningitis, Lassa, Guanarito, Junin, Machupo, Sabia, and Whitewater Arroyo viruses) and to provide determinants with the potential to be incorporated into a multivalent vaccine strategy.

Results

By inoculating HLA-DRB1*0101 transgenic mice with a panel of recombinant vaccinia viruses, each expressing a single arenavirus antigen, we identified 37 human HLA-DRB1*0101-restricted CD4⁺ T cell epitopes from the 7 antigenically distinct arenaviruses. We showed that the arenavirus-specific CD4⁺ T cell epitopes are capable of eliciting T cells with a propensity to provide help and protection through CD40L and polyfunctional cytokine expression. Importantly, we demonstrated that the set of identified CD4⁺ T cell epitopes provides broad, non-ethnically biased population coverage of all 7 arenavirus species targeted by our studies.

Conclusions

The identification of CD4⁺ T cell epitopes, with promiscuous binding properties, derived from 7 different arenavirus species will aid in the development of a T cell-based vaccine strategy with the potential to target a broad range of ethnicities within the general population and to protect against both Old and New World arenavirus infection.     

Morphological Comparison of Machupo with Lymphocytic Choriomeningitis Virus: Basis for a New Taxonomic Group

Striking morphologic similarities between Machupo, Tacaribe, and lymphocytic choriomeningitis viruses were found by thin-section electron microscopy. It is proposed that these viruses be brought together into a single taxonomic group.     

In silico characterization of immunogenic epitopes presented by HLA-Cw*0401

Background

Arenaviruses are a family of rodent-borne viruses that cause several hemorrhagic fevers. These diseases can be devastating and are often lethal. Herein, to aid in the design and development of diagnostics, treatments and vaccines for arenavirus infections, we have developed a database containing protein sequences from the seven pathogenic arenaviruses (Junin, Guanarito, Sabia, Machupo, Whitewater Arroyo, Lassa and LCMV).

Results

The database currently contains a non-redundant set of 333 protein sequences which were manually annotated. All entries were linked to NCBI and cited PubMed references. The database has a convenient query interface including BLAST search. Sequence variability analyses were also performed and the results are hosted in the database.

Conclusion

The database is available at http://epitope.liai.org:8080/projects/arena and can be used to aid in studies that require proteomic information from pathogenic arenaviruses.     

Highly Sensitive Assay for Measurement of Arenavirus-cell Attachment

Arenaviruses are a family of enveloped RNA viruses that cause severe human disease. The first step in the arenavirus life cycle is attachment of viral particles to host cells. While virus-cell attachment can be measured through the use of virions labeled with biotin, radioactive isotopes, or fluorescent dyes, these approaches typically require high multiplicities of infection (MOI) to enable detection of bound virus. We describe a quantitative (q)RT-PCR-based assay that measures Junin virus strain Candid 1 attachment via quantitation of virion-packaged viral genomic RNA. This assay has several advantages including its extreme sensitivity and ability to measure attachment over a large dynamic range of MOIs without the need to purify or label input virus. Importantly, this approach can be easily tailored for use with other viruses through the use of virus-specific qRT-PCR reagents. Further, this assay can be modified to permit measurement of particle endocytosis and genome uncoating. In conclusion, we describe a simple, yet robust assay for highly sensitive measurement of arenavirus-cell attachment.     

Selection for virulent dengue viruses occurs in humans and mosquitoes

Dengue is the most common mosquito-borne viral disease in humans. The spread of both mosquito vectors and viruses has led to the resurgence of epidemic dengue fever (a self-limited flu-like syndrome) and the emergence of dengue hemorrhagic fever (severe dengue with bleeding abnormalities) in urban centers of the tropics. There are no animal or laboratory models of dengue disease; indirect evidence suggests that dengue viruses differ in virulence, including their pathogenicities for humans and epidemic potential. We developed two assay systems (using human dendritic cells and Aedes aegypti mosquitoes) for measuring differences in virus replication that correlate with the potential to cause hemorrhagic dengue and increased virus transmission. Infection and growth experiments showed that dengue serotype 2 viruses causing dengue hemorrhagic fever epidemics (Southeast Asian genotype) can outcompete viruses that cause dengue fever only (American genotype). This fact implies that Southeast Asian genotype viruses will continue to displace other viruses, causing more hemorrhagic dengue epidemics.     

埃博拉出血热及埃博拉病毒的研究进展

埃博拉病毒可以引起一种人畜共患烈性传染病,即埃博拉出血热,此病于1976年始发于埃博拉河流域,并且于该区域严重流行,故而得名。人类一旦感染埃博拉病毒,死亡率可高达88%,从而引起医学界的广泛关注,世界卫生组织已将埃博拉病毒列为对人类危害最为严重的病毒之一。深入地了解埃博拉出血热及埃博拉病毒,及其致病机理,对于埃博拉出血热的预防和控制具有非常重要的意义。     

Rescue of a Recombinant Machupo Virus from Cloned cDNAs and In Vivo Characterization in Interferon (αβ/γ) Receptor Double Knockout Mice

Machupo virus (MACV) is the etiological agent of Bolivian hemorrhagic fever (BHF), a reemerging and neglected tropical disease associated with high mortality. The prototypical strain of MACV, Carvallo, was isolated from a human patient in 1963, but minimal in vitro and in vivo characterization has been reported. To this end, we utilized reverse genetics to rescue a pathogenic MACV from cloned cDNAs. The recombinant MACV (rMACV) had in vitro growth properties similar to those of the parental MACV. Both viruses caused similar disease development in alpha/beta and gamma interferon receptor knockout mice, including neurological disease development and high mortality. In addition, we have identified a novel murine model with mortality and neurological disease similar to BHF disease reported in humans and nonhuman primates.     

SAR studies of 4-acyl-1,6-dialkylpiperazin-2-one arenavirus cell entry inhibitors

Old World (Africa) and New World (South America) arenaviruses are associated with human hemorrhagic fevers. Efforts to develop small molecule therapeutics have yielded several chemical series including the 4-acyl-1,6-dialkylpiperazin-2-ones. Herein, we describe an extensive exploration of this chemotype. In initial Phase I studies, R¹ and R⁴ scanning libraries were assayed to identify potent substituents against Old World (Lassa) virus. In subsequent Phase II studies, R⁶ substituents and iterative R¹, R⁴ and R⁶ substituent combinations were evaluated to obtain compounds with improved Lassa and New World (Machupo, Junin, and Tacaribe) arenavirus inhibitory activity, in vitro human liver microsome metabolic stability and aqueous solubility.     

Do Canada geese (Branta canadensis Linnaeus, 1758) carry infectious agents for birds and man?

Currently, large groups of Canada geese (Branta canadensis Linnaeus, 1758) aggregate in recreational areas of north-western Germany. Questions have arisen as to whether these birds represent a special risk factor as a source of zoonotic agents for humans and as a source of viruses, causing notifiable or reportable diseases, for domestic poultry and waterfowl. To answer these questions, a total of 289 eggs were collected in 2002 and 2003 on a recreation site and assayed. Chlamydia psittaci was not isolated and neither was chlamydial antigen detected by polymerase chain reaction. All virus-isolation attempts were unsuccessful. Neither Salmonella spp. nor Campylobacter spp. was isolated from embryonic tissues, chorioallantoic membranes or yolk-sac membranes. The presence of antibodies against Newcastle disease virus and influenza A virus (haemagglutinin subtypes H5 and H7) was demonstrated in egg yolk. Antibodies were also detected against the egg-drop syndrome 1976 and duck plague viruses. It is concluded that further surveillance studies are needed for a reliable risk assessment.     

综述与专论: 树鼩病毒感染模型的研究新进展

病毒感染引起的疾病接近中国主要传染病发病率的一半,也是传染病致死的主要病因。建立与人类亲缘关系较近、方便有效的感染人类病毒的动物模型,对了解病毒的生物学特性、感染致病机理及制定有效防控措施具有重要意义。树鼩作为灵长类动物的近亲,与人类在生理生化、基因组学等方面的相似性远高于大鼠、小鼠等常用啮齿类实验动物,并具有个体小、便于实验操作、饲养成本低、能感染多种人类病毒等特点,作为动物模型在病毒感染性疾病研究中突显优势和潜能。本文从地区分布、进化、生物学特性等方面,阐述了树鼩作为动物模型应用于病毒感染性疾病研究的优势,包括在乙型肝炎病毒、甲型肝炎病毒,及其他病毒感染疾病研究中的进展。