Disparate Viruses Research Articles

Preparation and Evaluation of Recombinant Severe Fever with Thrombocytopenia Syndrome Virus Nucleocapsid Protein for Detection of Total Antibodies in Human and Animal Sera by Double-Antigen Sandwich Enzyme-Linked Immunosorbent Assay

The recent emergence of the human infection confirmed to be caused by severe fever with thrombocytopenia syndrome virus (SFTSV) in China is of global concern. Safe diagnostic immunoreagents for determination of human and animal seroprevalence in epidemiological investigations are urgently needed. This paper describes the cloning and expression of the nucleocapsid (N) protein of SFTSV. An N-protein-based double-antigen sandwich enzyme-linked immunosorbent assay (ELISA) system was set up to detect the total antibodies in human and animal sera. We reasoned that as the double-antigen sandwich ELISA detected total antibodies with a higher sensitivity than traditional indirect ELISA, it could be used to detect SFTSV-specific antibodies from different animal species. The serum neutralization test was used to validate the performance of this ELISA system. All human and animal sera that tested positive in the neutralization test were also positive in the sandwich ELISA, and there was a high correlation between serum neutralizing titers and ELISA readings. Cross-reactivity was evaluated, and the system was found to be highly specific to SFTSV; all hantavirus- and dengue virus-confirmed patient samples were negative. SFTSV-confirmed human and animal sera from both Anhui and Hubei Provinces in China reacted with N protein in this ELISA, suggesting no major antigenic variation between geographically disparate virus isolates and the suitability of this assay in nationwide application. ELISA results showed that 3.6% of the human serum samples and 47.7% of the animal field serum samples were positive for SFTSV antibodies, indicating that SFTSV has circulated widely in China. This assay, which is simple to operate, poses no biohazard risk, does not require sophisticated equipment, and can be used in disease surveillance programs, particularly in the screening of large numbers of samples from various animal species.

Protein glycosylation in infectious disease pathobiology and treatment.

A host of bacteria and viruses are dependent on O-linked and N-linked glycosylation to perform vital biological functions. Pathogens often have integral proteins that participate in host-cell interactions such as receptor binding and fusion with host membrane. Fusion proteins from a broad range of disparate viruses, such as paramyxovirus, HIV, ebola, and the influenza viruses share a variety of common features that are augmented by glycosylation. Each of these viruses contain multiple glycosylation sites that must be processed and modified by the host post-translational machinery to be fusogenically active. In most viruses, glycosylation plays a role in biogenesis, stability, antigenicity and infectivity. In bacteria, glycosylation events play an important role in the formation of flagellin and pili and are vitally important to adherence, attachment, infectivity and immune evasion. With the importance of glycosylation to pathogen survival, it is clear that a better understanding of the processes is needed to understand the pathogen requirement for glycosylation and to capitalize on this requirement for the development of novel therapeutics.

Identification of Essential Filovirion-associated Host Factors by Serial Proteomic Analysis and RNAi Screen

An assessment of the total protein composition of filovirus (ebolavirus and marburgvirus) virions is currently lacking. In this study, liquid chromatography-linked tandem mass spectrometry of purified ebola and marburg virions was performed to identify associated cellular proteins. Host proteins involved in cell adhesion, cytoskeleton, cell signaling, intracellular trafficking, membrane organization, and chaperones were identified. Significant overlap exists between this data set and proteomic studies of disparate viruses, including HIV-1 and influenza A, generated in multiple cell types. However, the great majority of proteins identified here have not been previously described to be incorporated within filovirus particles. Host proteins identified by liquid chromatography-linked tandem mass spectrometry could lack biological relevance because they represent protein contaminants in the virus preparation, or because they are incorporated within virions by chance. These issues were addressed using siRNA library-mediated gene knockdown (targeting each identified virion-associated host protein), followed by filovirus infection. Knockdown of several host proteins (e.g. HSPA5 and RPL18) significantly interfered with ebolavirus and marburgvirus infection, suggesting specific and relevant virion incorporation. Notably, select siRNAs inhibited ebolavirus, but enhanced marburgvirus infection, suggesting important differences between the two viruses. The proteomic analysis presented here contributes to a greater understanding of filovirus biology and potentially identifies host factors that can be targeted for antiviral drug development.

The Kaposi's sarcoma‐associated herpesvirus G protein‐coupled receptor: Lessons on dysregulated angiogenesis from a viral oncogene

Tumor viruses can induce cell transformation by overcoming cellular defense mechanisms and promoting the ungoverned proliferation of infected cells. To this end, functionally related viral oncogenes have evolved in disparate viruses to over-ride key proliferative and survival intracellular pathways, thus assuring efficient viral replication and contributing to tumor formation. Indeed, the study of viral oncogenes has been a powerful tool for disclosing fundamental insights into these basic cellular processes. In this regard, the Kaposi's sarcoma-associated herpesvirus (KSHV or HHV8), the etiological agent of Kaposi's sarcoma (KS), is an exemplary model of an oncogenic virus that includes within its genome several homologues of cellular genes implicated in the regulation of cell proliferation and apoptosis. However, emerging evidence now points to a single KSHV gene, ORF74, encoding for the viral G protein-coupled receptor (vGPCR), as essential for KS development. Expressed in only a fraction of cells within KS lesions, this viral receptor induces tumorigenesis through both autocrine and paracrine mechanisms. Indeed, work from several laboratories has demonstrated that vGPCR can promote cell proliferation, enhance cell survival, modulate cell migration, stimulate angiogenesis, and recruit inflammatory cells, both in expressing cells, as well as in neighboring (bystander) cells. Examination of this powerful viral oncogene may expose novel targets for the treatment of patients with KS and could ultimately provide a unique perspective into how GPCRs, and specifically chemokine receptors, contribute to angiogenesis and tumorigenesis.

Inducible Bronchus-Associated Lymphoid Tissue Elicited by a Protein Cage Nanoparticle Enhances Protection in Mice against Diverse Respiratory Viruses

BackgroundDestruction of the architectural and subsequently the functional integrity of the lung following pulmonary viral infections is attributable to both the extent of pathogen replication and to the host-generated inflammation associated with the recruitment of immune responses. The presence of antigenically disparate pulmonary viruses and the emergence of novel viruses assures the recurrence of lung damage with infection and resolution of each primary viral infection. Thus, there is a need to develop safe broad spectrum immunoprophylactic strategies capable of enhancing protective immune responses in the lung but which limits immune-mediated lung damage. The immunoprophylactic strategy described here utilizes a protein cage nanoparticle (PCN) to significantly accelerate clearance of diverse respiratory viruses after primary infection and also results in a host immune response that causes less lung damage.Methodology/Principal FindingsMice pre-treated with PCN, independent of any specific viral antigens, were protected against both sub-lethal and lethal doses of two different influenza viruses, a mouse-adapted SARS-coronavirus, or mouse pneumovirus. Treatment with PCN significantly increased survival and was marked by enhanced viral clearance, accelerated induction of viral-specific antibody production, and significant decreases in morbidity and lung damage. The enhanced protection appears to be dependent upon the prior development of inducible bronchus-associated lymphoid tissue (iBALT) in the lung in response to the PCN treatment and to be mediated through CD4+ T cell and B cell dependent mechanisms.Conclusions/SignificanceThe immunoprophylactic strategy described utilizes an infection-independent induction of naturally occurring iBALT prior to infection by a pulmonary viral pathogen. This strategy non-specifically enhances primary immunity to respiratory viruses and is not restricted by the antigen specificities inherent in typical vaccination strategies. PCN treatment is asymptomatic in its application and importantly, ameliorates the damaging inflammation normally associated with the recruitment of immune responses into the lung.

Darwinian selection for sites of Asn-linked glycosylation in phylogenetically disparate eukaryotes and viruses

Numerous protists and rare fungi have truncated Asn-linked glycan precursors and lack N-glycan-dependent quality control (QC) systems for glycoprotein folding in the endoplasmic reticulum. Here, we show that the abundance of sequons (NXT or NXS), which are sites for N-glycosylation of secreted and membrane proteins, varies by more than a factor of 4 among phylogenetically diverse eukaryotes, based on a few variables. There is positive correlation between the density of sequons and the AT content of coding regions, although no causality can be inferred. In contrast, there appears to be Darwinian selection for sequons containing Thr, but not Ser, in eukaryotes that have N-glycan-dependent QC systems. Selection for sequons with Thr, which nearly doubles the sequon density in human secreted and membrane proteins, occurs by an increased conditional probability that Asn and Thr are present in sequons rather than elsewhere. Increasing sequon densities of the hemagglutinin (HA) of influenza viruses A/H3N2 and A/H1N1 during the past few decades of human infection also result from an increased conditional probability that Asn, Thr, and Ser are present in sequons rather than elsewhere. In contrast, there is no selection on sequons by this mechanism in HA of A/H5N1 or 2009 A/H1N1 (Swine flu). Very strong selection for sequons with both Thr and Ser in glycoprotein of M(r) 120,000 (gp120) of HIV and related retroviruses results from this same mechanism, as well as amino acid composition bias and increases in AT content. We conclude that there is Darwinian selection for sequons in phylogenetically disparate eukaryotes and viruses.

Type I interferon-dependent regulation of granzyme B protein expression by memory CD8+ T cells during the early stages of a respiratory virus challenge (130.13)

Abstract Memory T cells established following the resolution of influenza and parainfluenza virus infections provide robust protection from a secondary virus challenge. Recently, we demonstrated that the rapid recruitment of circulating memory CD8+ T cells to the lung airways during the early stages of a secondary challenge is crucial for limiting early viral replication. In the course of these studies, we observed that the cytolytic protein granzyme B (gzmB) was highly expressed by virus-specific memory CD8+ T cells recruited to the lung airways. However, the mechanism by which gzmB protein is up-regulated in these cells was unclear. In the current study, we demonstrate that challenge with a disparate virus is sufficient to up-regulate the expression of gzmB protein, suggesting that this process is antigen-independent. To discern the mechanism of gzmB protein expression, we employed a mixed bone marrow chimera model to directly compare wild-type and gene-deficient virus-specific memory CD8+ T cells within the same host. These studies demonstrate that the antigen-independent expression of gzmB protein was dependent on STAT1, and requires signaling through the IFNα/β receptor. Thus, type I interferons act directly on memory CD8+ T cells to increase gzmB protein expression during the early stages of a secondary virus challenge. This work is supported by funds from the NIH and the Trudeau Institute.

Persistent Expression of Shrimp-virus Antigens in Two Insect Cell Lines Challenged with Two Shrimp Viruses

Experiments with crustacean viruses are hampered by lack of susceptible continuous cell lines. To overcome this problem, immortal mosquito and lepidopteran cell lines were both separately challenged with a shrimp DNA virus (white spot syndrome virus: WSSV, = PRDV) and RNA virus (yellow head virus: YHV) followed by serial, split-passage with immunohistochemical monitoring by confocal laser microscopy using labeled monoclonal antibodies to shrimp viral antigens. Stable, immortal cultures with 100% of the cells expressing shrimp-virus antigens were obtained, although the infected cells appeared grossly normal by phase contrast microscopy. Nor did they show any ultrastructural modifications characteristic of the challenge viruses. These persistently-expressing insect cell cultures were stable and could be continuously passaged, stored and revived as required. Since disparate viruses and insect cells were used, this appears to be a generic process that may be applicable to other shrimp viruses as well.

Benign circulation of rabbit haemorrhagic disease virus on Lambay Island, Eire

With the exception of virus strains Ashington and RCV, other recognised strains of Rabbit haemorrhagic disease virus (RHDV) share relatively close genetic homology. Using serology and phylogenetic analysis, we have identified a third disparate virus lineage in healthy rabbits on Lambay Island off the east coast of Eire, where disease due to RHDV has never been observed. ELISA tests revealed high titre RHDV-specific antibodies in 81% of the sera from 11 healthy rabbits captured on this island, indicating that the virus is actively circulating amongst these rabbits. Nevertheless, infectious virus has not been isolated from rabbits living on this island. The detection of antibodies and the disparate Lambay virus lineage in an apparently healthy and isolated wild rabbit population provides the most convincing evidence yet that at least some strains of RHDV can circulate harmlessly for long periods of time in wild rabbits possibly by producing persistent or latent infections.

A minimal kinetic model for a viral DNA packaging machine.

Terminase enzymes are common to both eukaryotic and prokaryotic double-stranded DNA viruses. These enzymes possess ATPase and nuclease activities that work in concert to "package" a viral genome into an empty procapsid, and it is likely that terminase enzymes from disparate viruses utilize a common packaging mechanism. Bacteriophage lambda terminase possesses a site-specific nuclease activity, a so-called helicase activity, a DNA translocase activity, and multiple ATPase catalytic sites that function to package viral DNA. Allosteric interactions between the multiple catalytic sites have been reported. This study probes these catalytic interactions using enzyme kinetic, photoaffinity labeling, and vanadate inhibition studies. The ensemble of data forms the basis for a minimal kinetic model for lambda terminase. The model incorporates an ADP-driven conformational reorganization of the terminase subunits assembled on viral DNA, which is central to the activation of a catalytically competent packaging machine. The proposed model provides a unifying mechanism for allosteric interaction between the multiple catalytic sites of the holoenzyme and explains much of the kinetic data in the literature. Given that similar packaging mechanisms have been proposed for viruses as dissimilar as lambda and the herpes viruses, the model may find general utility in our global understanding of the enzymology of virus assembly.

Polydnavirus integration in lepidopteran host cells in vitro

The long-term persistence of polydnavirus (PDV) DNA in infected lepidopteran cell cultures has suggested that at least some of the virus sequences become integrated permanently into the cell genome. In the current study, we provide supportive evidence of this event. Cloned libraries were prepared from two different Lymantria dispar (gypsy moth) cell lines that had been maintained in continuous culture for more than five years after infection with Glyptapanteles indiensis PDV (GiPDV). Junction clones containing both insect chromosomal and polydnaviral sequences were isolated. Precise integration junction sites were identified by sequence comparison of linear (integrated) and circular forms of the GiPDV genome segment F, from which viral sequences originated. Host chromosomal sequences at the site of integration varied between the two L. dispar cell lines but virus sequence junctions were identical and contained a 4-base pair CATG palindromic repeat. The GiPDV segment F does not encode any self-replication or self-insertion proteins, suggesting a host-derived mechanism is responsible for its in vitro integration. The chromosomal site of one junction clone contained sequences indicative of a new L. dispar retrotransposon, including a putative reverse transcriptase and integrase located upstream of the site of viral integration. A potential mechanism is proposed for the integration of PDV DNA in vitro. It remains to be seen if integration of the virus also occurs in the lepidopteran host in vivo.

The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor.

Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein. Structural models of the transmembrane glycoproteins (GP-2) of the Arenaviruses, lymphochoriomeningitis virus (LCMV) and Lassa fever virus, are presented, based on consistent structural propensities despite variation in the amino acid sequence. The principal features of the model, a hydrophobic amino terminus, and two antiparallel helices separated by a glycosylated, antigenic apex, are common to a number of otherwise disparate families of enveloped RNA viruses. Within the first amphipathic helix, demonstrable by circular dichroism of a peptide fragment, there is a highly conserved heptad repeat pattern proposed to mediate multimerization by coiled-coil interactions. The amino terminal 18 amino acids are 28% identical and 50% highly similar to the corresponding region of Ebola, a member of the Filovirus family. Within the second, charged helix just prior to membrane insertion there is also high similarity over the central 18 amino acids in corresponding regions of Lassa and Ebola, which may be further related to the similar region of HIV-1 defining a potent antiviral peptide analogue. These findings indicate a common pattern of structure and function among viral transmembrane fusion proteins from a number of virus families. Such a pattern may define a viral transmembrane superfamily that evolved from a common precursor eons ago.

Virus Membrane Fusion Proteins: Biological Machines that Undergo a Metamorphosis

Fusion proteins from a group of widely disparate viruses, including the paramyxovirus F protein, the HIV and SIV gp160 proteins, the retroviral Env protein, the Ebola virus Gp, and the influenza virus haemagglutinin, share a number of common features. All contain multiple glycosylation sites, and must be trimeric and undergo proteolytic cleavage to be fusogenically active. Subsequent to proteolytic cleavage, the subunit containing the transmembrane domain in each case has an extremely hydrophobic region, termed the fusion peptide, or at near its newly generated N-terminus. In addition, all of these viral fusion proteins have 4-3 heptad repeat sequences near both the fusion peptide and the transmembrane domain. These regions have been demonstrated from a tight complex, in which the N-terminal heptad repeat forms a trimeric-coiled coil, with the C-terminal heptad repeat forming helical regions that buttress the coiled-coil in an anti-parallel manner. The significance of each of these structural elements in the processing and function of these viral fusion proteins is discussed.

The paramyxovirus fusion protein forms an extremely stable core trimer: structural parallels to influenza virus haemagglutinin and HIV-1 gp41.

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit F2 and a membrane anchored subunit F1. A highly stable structure has been identified comprised of peptides derived from the simian virus 5 (SV5) F1 heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the SV5 F1 heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% alpha-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm > 90 degrees C), 82% alpha-helical, discrete trimer of heterodimers (mass 31,300 M(r)) that is resistant to denaturation by 2% SDS at 40 degrees C. The authors suggest that this alpha-helical trimeric complex represents the core most stable form of the F protein that is either fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptide N-1 inhibits cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41 and influenza virus haemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.

A Core Trimer of the Paramyxovirus Fusion Protein: Parallels to Influenza Virus Hemagglutinin and HIV-1 gp41

The paramyxovirus fusion (F) protein mediates membrane fusion. The biologically active F protein consists of a membrane distal subunit, F2, and a membrane-anchored subunit, F1. We have identified a highly stable structure composed of peptides derived from the F1heptad repeat A, which abuts the hydrophobic fusion peptide (peptide N-1), and the F1heptad repeat B, located 270 residues downstream and adjacent to the transmembrane domain (peptides C-1 and C-2). In isolation, peptide N-1 is 47% α-helical and peptide C-1 and C-2 are unfolded. When mixed together, peptides N1 + C1 form a thermostable (Tm>90°C), 82% α-helical, discrete trimer of heterodimers (mass 31,300Mr) that is resistant to denaturation by 2% SDS at 40°C. We suggest that this α-helical trimeric complex represents the core most stable form of the F protein that either is fusion competent or forms after fusion has occurred. Peptide C-1 is a potent inhibitor of both the lipid mixing and the aqueous content mixing fusion activity of the SV5 F protein. In contrast, peptides N-1 and N-2 inhibit cytoplasmic content mixing but not lipid mixing, leading to a stable hemifusion state. Thus, these peptides define functionally different steps in the fusion process. The parallels among both the fusion processes and the protein structures of paramyxovirus F proteins, HIV gp41, and influenza virus hemagglutinin are discussed, as the analogies are indicative of a conserved paradigm for fusion promotion among fusion proteins from widely disparate viruses.

Phylogenetic analysis of pestiviruses from domestic and wild ruminants.

Infections with pestiviruses occur in cattle, sheep, pigs and also in numerous other ungulate species. In the present study, pestiviruses from goat, buffalo, deer and giraffe were analysed at the molecular level; unusual strains from cattle and pigs were also included. A phylogenetic analysis of the respective pestiviruses was undertaken on the basis of a fragment from the 5' noncoding region as well as the gene encoding autoprotease Npro. Statistical analyses of the respective phylogenetic trees-based on the 5' NCR revealed low confidence levels for most of the branches, while the structure of the tree based on the Npro gene was supported by high bootstrap values. Accordingly, the isolates from goat, buffalo and deer can be grouped together with bovine viral diarrhoea virus (pestivirus type 1); within this genotype three subgroups and one disparate virus have been identified. One isolate from pig and one from cattle belong to the group of 'true' border disease virus (pestivirus type 3), which can be further subdivided into two major subgroups. Interestingly, the giraffe isolate does not belong to one of the four established pestivirus genotypes. The phylogenetic analysis strongly suggests that genotype 1 pestiviruses occur world-wide in many ruminant species. Furthermore, phylogenetic trees based on the Npro gene nucleotide sequences show that the respective sequences do not segregate into discrete lineages based on host-species origin.

Rapid identification of Australian bunyavirus isolates belonging to the Simbu serogroup using indirect ELISA formats

The Bunyavirus genus, belonging to the Bunyaviridae family, is comprised of a large group of antigenically and geographically disparate arthropod-borne viruses of medical and veterinary significance. In Australia, viruses belonging to the Simbu serogroup of the Bunyavirus genus, Akabane, Tinaroo, Peaton, Aino, Douglas, Thimiri and Facey's Paddock have been isolated. In this communication we describe two indirect ELISAs, referred to as the Simbu serogroup ELISA (SG-ELISA), and the Simbu typing ELISA (ST-ELISA), for the identification of these Simbu serogroup viruses. Infected cell lysate antigens prepared from Simbu serogroup virus isolates were assessed in the SG-ELISA for reactivity with a mouse monoclonal antibody (4H9/B11/F1). The monoclonal antibody reacted strongly with all Australian members of Simbu serogroup reference viruses and is proposed for use as a serogrouping reagent for Simbu viruses. Furthermore, the ST-ELISA enabled specific identification of viruses from within this group by recognition of characteristic reaction patterns between infected cell lysate antigens and a panel of polyclonal antisera raised to Simbu serogroup viruses.

Influenza B virus PB1 protein: Nucleotide sequence of the genome RNA segment predicts a high degree of structural homology with the corresponding influenza A virus polymerase protein

The complete nucleotide sequence of a cloned cDNA copy of the genome RNA segment encoding the influenza B/Lee/40 virus PB1 polymerase protein has been determined. The genome RNA segment is 2368 nucleotides in length and is capable of encoding a polymerase (PBl) protein of 752 amino acids with a calculated mol mass of 84,407 Da. As expected, the protein is highly basic with a net charge of +20 at pH 7.0. Sequence comparison between the influenza A and B virus PB1 proteins reveals that they share 61% amino acid homology. An internal hydrophobic domain and 90% of the proline residues found within the influenza A virus PB1 protein are conserved in the influenza B virus molecule. The influenza A and B virus PB1 proteins share the highest homology yet seen between proteins encoded by these disparate viruses. This remarkable conservation of primary structure argues for severe functional constraint on the evolution of this influenza virus polymerase protein.

USE OF SERIAL SECTIONS TO DELINEATE THE STRUCTURE OF PORTHETRIA DISPAR VIRUS IN THE ELECTRON MICROSCOPE

Consecutive serial sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus revealed bundles of viral rods scattered and oriented at random within the polyhedral body. Each bundle was entirely surrounded by a dense, sharply defined membrane. The rods measured 18 to 22 mmicro in diameter and averaged 280 mmicro in length. No spherical viral particles were encountered. The effects of variable compression and periodic distortion of the sections on the appearance of the virus are discussed.

The macromolecular paracrystalline lattice of insect viral polyhedral bodies demonstrated in ultrathin sections examined in the electron microscope.

Thin sections of polyhedra obtained from gipsy moth larvae infected with P. dispar virus and from silkworm larvae infected with B. mori virus revealed viral particles contained within a pseudohexagonal, macromolecular, paracrystalline lattice. The gipsy moth virus occurs in bundles of one to eight rods enclosed by a limiting membrane. The particles of the silkworm virus, although generally occurring singly, also possess a limiting membrane. The macromolecules appear to be dense, discrete particles when cross-sectioned and to form dense bands by superimposition when longitudinally or obliquely sectioned at certain angles. Calculations of macromolecular size have been made.