Showing papers in "Virology Journal in 2007"
TL;DR: The results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role.
Abstract: Background
Nipah virus (NiV) is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs).
203 citations
TL;DR: It is suggested that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemag GLUTinin density on the cell surface, and the presence of high levels of α2-6 sialic acids on a cell surface does not guarantee productive replication of a virus with α 2-6 receptor specificity.
Abstract: Human influenza viruses are known to bind to sialic acid linked α2-6 to galactose, but the binding specificity beyond that linkage has not been systematically examined. H3N2 human influenza isolates lost binding to chicken red cells in the 1990s but viruses isolated since 2003 have re-acquired the ability to agglutinate chicken erythrocytes. We have investigated specificity of binding, changes in hemagglutinin sequence of the recent viruses and the role of sialic acid in productive infection. Viruses that agglutinate, or do not agglutinate, chicken red cells show identical binding to a Glycan Array of 264 oligosaccharides, binding exclusively to a subset of α2-6-sialylsaccharides. We identified an amino acid change in hemagglutinin that seemed to correlate with chicken red cell binding but when tested by mutagenesis there was no effect. Recombinant hemagglutinins expressed on Sf-9 cells bound chicken red cells but the released recombinant baculoviruses agglutinated only human red cells. Similarly, an isolate that does not agglutinate chicken red cells show hemadsorption of chicken red cells to infected MDCK cells. We suggest that binding of chicken red cells to cell surface hemagglutinin but not to virions is due to a more favorable hemagglutinin density on the cell surface. We investigated whether a virus specific for α2-6 sialyloligosaccharides shows differential entry into cells that have varying proportions of α2-6 and α2-3 sialic acids, including human A549 and HeLa cells with high levels of α2-6 sialic acid, and CHO cells that have only α2-3 sialic acid. We found that the virus enters all cell types tested and synthesizes viral nucleoprotein, localized in the nucleus, and hemagglutinin, transported to the cell surface, but infectious progeny viruses were released only from MDCK cells. Agglutination of chicken red cells does not correlate with altered binding to any oligosaccharide on the Glycan Array, and may result from increased avidity due to density of hemagglutinin and not increased affinity. Absence of α2-6 sialic acid does not protect a cell from influenza infection and the presence of high levels of α2-6-sialic acids on a cell surface does not guarantee productive replication of a virus with α2-6 receptor specificity.
174 citations
TL;DR: The re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses, and further re-defining the core set of iridovirus genes will continue to lead to a better understanding of the phylogenetic relationships between individual irids.
Abstract: Members of the family Iridoviridae can cause severe diseases resulting in significant economic and environmental losses. Very little is known about how iridoviruses cause disease in their host. In the present study, we describe the re-analysis of the Iridoviridae family of complex DNA viruses using a variety of comparative genomic tools to yield a greater consensus among the annotated sequences of its members. A series of genomic sequence comparisons were made among, and between the Ranavirus and Megalocytivirus genera in order to identify novel conserved ORFs. Of these two genera, the Megalocytivirus genomes required the greatest number of altered annotations. Prior to our re-analysis, the Megalocytivirus species orange-spotted grouper iridovirus and rock bream iridovirus shared 99% sequence identity, but only 82 out of 118 potential ORFs were annotated; in contrast, we predict that these species share an identical complement of genes. These annotation changes allowed the redefinition of the group of core genes shared by all iridoviruses. Seven new core genes were identified, bringing the total number to 26. Our re-analysis of genomes within the Iridoviridae family provides a unifying framework to understand the biology of these viruses. Further re-defining the core set of iridovirus genes will continue to lead us to a better understanding of the phylogenetic relationships between individual iridoviruses as well as giving us a much deeper understanding of iridovirus replication. In addition, this analysis will provide a better framework for characterizing and annotating currently unclassified iridoviruses.
165 citations
TL;DR: It is proposed that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition, resulting in failure to enter mitosis.
Abstract: HIV-1 Vpr is a viral accessory protein that activates ATR through the induction of DNA replication stress. ATR activation results in cell cycle arrest in G2 and induction of apoptosis. In the present study, we investigate the role of the ubiquitin/proteasome system (UPS) in the above activity of Vpr. We report that the general function of the UPS is required for Vpr to induce G2 checkpoint activation, as incubation of Vpr-expressing cells with proteasome inhibitors abolishes this effect. We further investigated in detail the specific E3 ubiquitin ligase subunits that Vpr manipulates. We found that Vpr binds to the DCAF1 subunit of a cullin 4a/DDB1 E3 ubiquitin ligase. The carboxy-terminal domain Vpr(R80A) mutant, which is able to bind DCAF1, is inactive in checkpoint activation and has dominant-negative character. In contrast, the mutation Q65R, in the leucine-rich domain of Vpr that mediates DCAF1 binding, results in an inactive Vpr devoid of dominant negative behavior. Thus, the interaction of Vpr with DCAF1 is required, but not sufficient, for Vpr to cause G2 arrest. We propose that Vpr recruits, through its carboxy terminal domain, an unknown cellular factor that is required for G2-to-M transition. Recruitment of this factor leads to its ubiquitination and degradation, resulting in failure to enter mitosis.
155 citations
TL;DR: Evidence is provided that Dengue infection induces and regulates the three branches of the UPR signaling cascades, a basis for the understanding of the viral regulation and conditions beneficial to the viral infection.
Abstract: Dengue and Dengue hemorrhagic fever have emerged as some of the most important mosquito-borne viral diseases in the tropics. The mechanisms of pathogenesis of Dengue remain elusive. Recently, virus-induced apoptosis mediated by the Unfolded Protein Response (UPR) has been hypothesised to represent a crucial pathogenic event in viral infection. In an attempt to evaluate the contribution of the UPR to virus replication, we have characterized each component of this signalling pathway following Dengue virus infection. We find that upon Dengue virus infection, A549 cells elicit an UPR which is observed at the level of translation attenuation (as visualized by the phosphorylation of eIF2alpha) and activation of specific pathways such as nuclear translocation of ATF-6 and splicing of XBP-1. Interestingly, we find that specific serotype of virus modulate the UPR with different selectivity. In addition, we demonstrate that perturbation of the UPR by preventing the dephosphorylation of the translation initiation factor eIF2alpha using Salubrinal considerably alters virus infectivity. This report provides evidence that Dengue infection induces and regulates the three branches of the UPR signaling cascades. This is a basis for our understanding of the viral regulation and conditions beneficial to the viral infection. Furthermore, modulators of UPR such as Salubrinal that inhibit Dengue replication may open up an avenue toward cell-protective agents that target the endoplasmic reticulum for anti-viral therapy.
144 citations
TL;DR: The state of the art of AAV-based vectors studies and the advances on the use of A AV vectors for several gene therapy approaches are highlighted.
Abstract: The adeno-associated virus (AAV) has rapidly gained popularity in gene therapy since the establishment of the first AAV2 infectious clone, in 1982, due to some of their distinguishing characteristics such as lack of pathogenicity, wide range of infectivity, and ability to establish long-term transgene expression. Notably over the past decade, this virus has attracted considerable interest as a gene therapy vector, and about 85% of the currently available 2,041 PubMed references on adeno-associated viruses have been published during this time. The exponential progress of AAV-based vectors has been made possible by the advances in the knowledge of the virology and biology of this virus, which allows great improvement in AAV vectors construction and a better comprehension of their operation. Moreover, with the recent discovery of novel AAV serotypes, there is virtually one preferred serotype for nearly every organ or tissue to target. Thus, AAV-based vectors have been successfully overcoming the main gene therapy challenges such as transgene maintenance, safety and host immune response, and meeting the desirable vector system features of high level of safety combined with clinical efficacy and versatility in terms of potential applications. Consequently, AAV is increasingly becoming the vector of choice for a wide range of gene therapy approaches. This report will highlight the state of the art of AAV-based vectors studies and the advances on the use of AAV vectors for several gene therapy approaches.
134 citations
TL;DR: Age, parity, gestational age, residence, history of blood transfusion, dental manipulations, tattooing and circumcision did not contribute significantly to increased HBVsAg sero-positivity and there was low prevalence of Anti-HCV.
Abstract: Background
The epidemiology of viral hepatitis during pregnancy is essential for health planners and programme managers. While much data exist concerning viral hepatitis during pregnancy in many African countries, no proper published data are available in Sudan.
133 citations
TL;DR: Although all 4 viruses tested share high genetic similarity in all 8 genes, only the Ohio strain was shown to transmit efficiently both ways between swine and turkeys, and one isolate was able to transmit from pigs to turkeys but not vice versa.
Abstract: The triple reassortant H3N2 viruses were isolated for the first time from pigs in 1998 and are known to be endemic in swine and turkey populations in the United States. In 2004, we isolated two H3N2 triple reassortant viruses from two turkey breeder flocks in Ohio and Illinois. Infected hens showed no clinical signs, but experienced a complete cessation of egg production. In this study, we evaluated three triple reassortant H3N2 isolates of turkey origin and one isolate of swine origin for their transmission between swine and turkeys. Although all 4 viruses tested share high genetic similarity in all 8 genes, only the Ohio strain (A/turkey/Ohio/313053/04) was shown to transmit efficiently both ways between swine and turkeys. One isolate, A/turkey/North Carolina/03, was able to transmit from pigs to turkeys but not vice versa. Neither of the other two viruses transmitted either way. Sequence analysis of the HA1 gene of the Ohio strain showed one amino acid change (D to A) at residue 190 of the receptor binding domain upon transmission from turkeys to pigs. The Ohio virus was then tested for intraspecies transmission in three different avian species. The virus was shown to replicate and transmit among turkeys, replicate but does not transmit among chickens, and did not replicate in ducks. Identifying viruses with varying inter- and intra-species transmission potential should be useful for further studies on the molecular basis of interspecies transmission.
126 citations
TL;DR: Using RT-PCR, sequences of a genetically distinct hantavirus, designated Seewis virus (SWSV), were amplified from lung tissue of a Eurasian common shrew, captured in October 2006 in Graubünden, Switzerland.
Abstract: More than 20 years ago, hantaviral antigens were reported in tissues of the Eurasian common shrew (Sorex araneus), Eurasian water shrew (Neomys fodiens) and common mole (Talpa europea), suggesting that insectivores, or soricomorphs, might serve as reservoirs of unique hantaviruses. Using RT-PCR, sequences of a genetically distinct hantavirus, designated Seewis virus (SWSV), were amplified from lung tissue of a Eurasian common shrew, captured in October 2006 in Graubunden, Switzerland. Pair-wise analysis of the full-length S and partial M and L segments of SWSV indicated approximately 55%–72% similarity with hantaviruses harbored by Murinae, Arvicolinae, Neotominae and Sigmodontinae rodents. Phylogenetically, SWSV grouped with other recently identified shrew-borne hantaviruses. Intensified efforts are underway to clarify the genetic diversity of SWSV throughout the geographic range of the Eurasian common shrew, as well as to determine its relevance to human health.
111 citations
TL;DR: It is demonstrated that both NLS1 and NLS2 on NP can mediate the nuclear uptake of influenza A vRNPs, and when both sequences were blocked, vRNP nuclear import was almost completely inhibited.
Abstract: The influenza A virus replicates in the nucleus of its host cell. Thus, entry of the influenza genome into the cell nucleus is necessary for establishing infection. The genome of the influenza A virus consists of eight single-stranded, negative-sense RNA molecules, individually packed with several copies of the viral nucleoprotein (NP) into ribonucleoprotein particles (vRNPs). These vRNPs are large, rod-shaped complexes containing a core of NP, around which the RNA is helically wrapped. The vRNPs are the entities that enter the nucleus, and their nuclear import must be mediated by nuclear localization sequences (NLSs) exposed on the vRNPs. NP contains at least two putative NLSs, one at the N-terminus (NLS1) and one in the middle (NLS2) of the protein. These NP NLSs have been shown to mediate the nuclear import of recombinant NP molecules. However, it remains to be determined which NLS mediates the nuclear import of influenza vRNP complexes. To directly track the nuclear import of the influenza A genome, we developed an experimental assay based on digitonin-permeabilized cells and fluorescently-labeled vRNPs isolated from the influenza A virus. We used this assay to determine the contribution of the two proposed NLSs on NP to the nuclear import of influenza vRNP complexes. Peptides that mimic each of the two NLSs on NP were used to compete with vRNPs for their nuclear import receptors. In addition, antibodies against the two NP NLSs were used to block the NLSs on the vRNP complexes, and thereby inhibit vRNP nuclear import. Both peptide competition and antibody inhibition of either sequence resulted in decreased nuclear accumulation of vRNPs. The two sequences act independently of each other, as inhibition of only one of the two NLSs still resulted in significant, though diminished, nuclear import of vRNPs. Furthermore, when both sequences were blocked, vRNP nuclear import was almost completely inhibited. Antibody inhibition studies further showed that NLS1 on NP is the main contributor to the nuclear import of vRNPs. Our results demonstrate that both NLS1 and NLS2 on NP can mediate the nuclear uptake of influenza A vRNPs.
108 citations
TL;DR: In this article, the authors used propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology.
Abstract: The number of successful propagations/isolations of soil-borne bacteriophages is small in comparison to the number of bacteriophages observed by microscopy (great plaque count anomaly). As one resolution of the great plaque count anomaly, we use propagation in ultra-dilute agarose gels to isolate a Bacillus thuringiensis bacteriophage with a large head (95 nm in diameter), tail (486 × 26 nm), corkscrew-like tail fibers (187 × 10 nm) and genome (221 Kb) that cannot be detected by the usual procedures of microbiology. This new bacteriophage, called 0305φ8-36 (first number is month/year of isolation; remaining two numbers identify the host and bacteriophage), has a high dependence of plaque size on the concentration of a supporting agarose gel. Bacteriophage 0305φ8-36 does not propagate in the traditional gels used for bacteriophage plaque formation and also does not produce visible lysis of liquid cultures. Bacteriophage 0305φ8-36 aggregates and, during de novo isolation from the environment, is likely to be invisible to procedures of physical detection that use either filtration or centrifugal pelleting to remove bacteria. Bacteriophage 0305φ8-36 is in a new genomic class, based on genes for both structural components and DNA packaging ATPase. Thus, knowledge of environmental virus diversity is expanded with prospect of greater future expansion.
TL;DR: It is suggested that CD151 should cooperate in PRRSV infection in vitro in MARC-145 and BHK-21 cells.
Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA virus causing respiratory and reproductive diseases in swine. The susceptibility for PRRSV varies between the different breeds of swine. In cell culture, PRRSV virus can be propagated in primary porcine alveolar macrophages and some African green monkey kidney cell lines, such as MARC-145 cells. Previous studies have shown that 3' untranslated region (UTR) RNAs of the arteriviruses play an important role in the replication of the virus through interactions with cellular proteins. To better understand the differences in the replication capability of PRRSV in different cell lines, we sought to identify the host cellular proteins interacting with PRRSV 3' UTR RNA. We constructed a cDNA library of MARC-145 cell line in lambda ZAP Express vector and screened the library with the positive sense 3' UTR RNA of PRRSV. We found that CD151, a host cellular protein, interacting with PRRSV 3' UTR RNA. The specificity of the interaction between CD151 and PRRSV 3' UTR RNA was examined by gel shift assay as well as North-Western hybridization. The transfection of CD151 expression clone into BHK-21 rendered these cells susceptible to PRRSV infection, and the transfection of siRNA against CD151 into MARC-145 significantly reduced the level of PRRSV infection. Also, anti-CD151 antibody treatment to MARC-145 completely blocked PRRSV infection. Based on our results, we suggest that CD151 should cooperate in PRRSV infection in vitro in MARC-145 and BHK-21 cells.
TL;DR: The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress.
Abstract: The VP2 outer capsid protein Bluetongue Virus (BTV) is responsible for receptor binding, haemagglutination and eliciting host-specific immunity. However, the assembly of this outer capsid protein on the transcriptionally active viral core would block transcription of the virus. Thus assembly of the outer capsid on the core particle must be a tightly controlled process during virus maturation. Earlier studies have detected mature virus particles associated with intermediate filaments in virus infected cells but the viral determinant for this association and the effect of disrupting intermediate filaments on virus assembly and release are unknown. In this study it is demonstrated that BTV VP2 associates with vimentin in both virus infected cells and in the absence of other viral proteins. Further, the determinants of vimentin localisation are mapped to the N-terminus of the protein and deletions of aminio acids between residues 65 and 114 are shown to disrupt VP2-vimentin association. Site directed mutation also reveals that amino acid residues Gly 70 and Val 72 are important in the VP2-vimentin association. Mutation of these amino acids resulted in a soluble VP2 capable of forming trimeric structures similar to unmodified protein that no longer associated with vimentin. Furthermore, pharmacological disruption of intermediate filaments, either directly or indirectly through the disruption of the microtubule network, inhibited virus release from BTV infected cells. The principal findings of the research are that the association of mature BTV particles with intermediate filaments are driven by the interaction of VP2 with vimentin and that this interaction contributes to virus egress. Furthermore, i) the N-terminal 118 amino acids of VP2 are sufficient to confer vimentin interaction. ii) Deletion of amino acids 65–114 or mutation of amino acids 70–72 to DVD abrogates vimentin association. iii) Finally, disruption of vimentin structures results in an increase in cell associated BTV and a reduction in the amount of released virus from infected cells.
TL;DR: A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection, and the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4.
Abstract: Rotavirus is the major cause of infantile gastroenteritis and each year causes 611 000 deaths worldwide. The virus infects the mature enterocytes of the villus tip of the small intestine and induces a watery diarrhea. Diarrhea can occur with no visible tissue damage and, conversely, the histological lesions can be asymptomatic. Rotavirus impairs activities of intestinal disaccharidases and Na+-solute symports coupled with water transport. Maldigestion of carbohydrates and their accumulation in the intestinal lumen as well as malabsorption of nutrients and a concomitant inhibition of water reabsorption can lead to a malabsorption component of diarrhea. Since the discovery of the NSP4 enterotoxin, diverse hypotheses have been proposed in favor of an additional secretion component in the pathogenesis of diarrhea. Rotavirus induces a moderate net chloride secretion at the onset of diarrhea, but the mechanisms appear to be quite different from those used by bacterial enterotoxins that cause pure secretory diarrhea. Rotavirus failed to stimulate Cl- secretion in crypt, whereas it stimulated Cl- reabsorption in villi, questioning, therefore, the origin of net Cl- secretion. A solution to this riddle was that intestinal villi do in fact secrete chloride as a result of rotavirus infection. Also, the overall chloride secretory response is regulated by a phospholipase C-dependent calcium signaling pathway induced by NSP4. However, the overall response is weak, suggesting that NSP4 may exert both secretory and subsequent anti-secretory actions, as did carbachol, hence limiting Cl- secretion. All these characteristics provide the means to make the necessary functional distinction between viral NSP4 and bacterial enterotoxins.
TL;DR: The most recent outbreak of RHDV in the USA occurred in the state of Indiana in June of 2005 as mentioned in this paper, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages.
Abstract: Rabbit Hemorrhagic Disease (RHD) is a severe acute viral disease specifically affecting the European rabbit Oryctolagus cuniculus. As the European rabbit is the predominant species of domestic rabbit throughout the world, RHD contributes towards significant losses to rabbit farming industries and endangers wild populations of rabbits in Europe and other predatory animals in Europe that depend upon rabbits as a food source. Rabbit Hemorrhagic Disease virus (RHDV) – a Lagovirus belonging to the family Caliciviridae is the etiological agent of RHD. Typically, RHD presents with sudden death in 70% to 95% of infected animals. There have been four separate incursions of RHDV in the USA, the most recent of which occurred in the state of Indiana in June of 2005. Animal inoculation studies confirmed the pathogenicity of the Indiana 2005 isolate, which caused acute death and pathological changes characterized by acute diffuse severe liver necrosis and pulmonary hemorrhages. Complete viral genome sequences of all USA outbreak isolates were determined and comparative genomics revealed that each outbreak was the result of a separate introduction of virus rather than from a single virus lineage. All of the USA isolates clustered with RHDV genomes from China, and phylogenetic analysis of the major capsid protein (VP60) revealed that they were related to a pandemic antigenic variant strain known as RHDVa. Rapid spread of the RHDVa pandemic suggests a selective advantage for this new subtype. Given its rapid spread, pathogenic nature, and potential to further evolve, possibly broadening its host range to include other genera native to the Americas, RHDVa should be regarded as a threat.
TL;DR: This review suggests that this PB1-F2 protein, present in all previous pandemic strains, may reappear as a virulence factor in a subsequent pandemic strain.
Abstract: With the prospect of another pandemic Influenza fresh in our consciousness, the pathogenic nature of the Influenza A virus and its ability to induce high rates of mortality are ever more pertinent. Recently a novel protein encoded by an alternate reading frame in the PB1 Gene segment of Influenza A virus has been discovered and in turn shown to enhance viral virulence in a mouse model [1]. This protein has been shown to specifically target and destroy alveolar macrophages [2]. This review suggests that this protein, present in all previous pandemic strains, may reappear as a virulence factor in a subsequent pandemic strain. This PB1-F2 protein will enhance the mortality rate of the virus by increasing the likelihood of a secondary bacterial infection, which is the primary cause of death to a patient infected with Influenza A.
TL;DR: This work generates a representative set of 34 fully sequenced HRVs and reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution.
Abstract: The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution.
TL;DR: It is imperative that what is known about the molecular biology of this pathogen and the intricacies of its interaction with its host are critically evaluated so that in future attempts to more effectively combat this important human pathogen.
Abstract: Poliomyelitis has afflicted humankind since antiquity, and for nearly a century now, we have known the causative agent, poliovirus. This pathogen is an enterovirus that in recent history has been the source of a great deal of human suffering. Although comparatively small, its genome is packed with sufficient information to make it a formidable pathogen. In the last 20 years the Global Polio Eradication Initiative has proven successful in greatly diminishing the number of cases worldwide but has encountered obstacles in its path which have made halting the transmission of wild polioviruses a practical impossibility. As we begin to realize that a change in strategy may be crucial in achieving success in this venture, it is imperative that we critically evaluate what is known about the molecular biology of this pathogen and the intricacies of its interaction with its host so that in future attempts we may better equipped to more effectively combat this important human pathogen.
TL;DR: The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005, which may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time.
Abstract: Since 2005 highly pathogenic (HP) avian influenza A H5N1 viruses have spread from Asia to Africa and Europe infecting poultry, humans and wild birds HP H5N1 virus was isolated in Denmark for the first time in March 2006 A total of 44 wild birds were found positive for the HP H5N1 infection In addition, one case was reported in a backyard poultry flock Full-genome characterisation of nine isolates revealed that the Danish H5N1 viruses were highly similar to German H5N1 isolates in all genes from the same time period The haemagglutinin gene grouped phylogenetically in H5 clade 2 subclade 2 and closest relatives besides the German isolates were isolates from Croatia in 2005, Nigeria and Niger in 2006 and isolates from Astrakhan in Russia 2006 The German and Danish isolates shared unique substitutions in the NA, PB1 and NS2 proteins The first case of HP H5N1 infection of wild and domestic birds in Denmark was experienced in March 2006 This is the first full genome characterisation of HP H5N1 avian influenza A virus in the Nordic countries The Danish viruses from this time period have their origin from the wild bird strains from Qinghai in 2005 These viruses may have been introduced to the Northern Europe through unusual migration due to the cold weather in Eastern Europe at that time
TL;DR: Information is gathered on TM in an attempt to shed some light on the functional regions of this protein, which is not well understood and is fiercely debated.
Abstract: The human immunodeficiency virus type 1 (HIV-1) has been intensely investigated since its discovery in 1983 as the cause of acquired immune deficiency syndrome (AIDS). With relatively few proteins made by the virus, it is able to accomplish many tasks, with each protein serving multiple functions. The Envelope glycoprotein, composed of the two noncovalently linked subunits, SU (surface glycoprotein) and TM (transmembrane glycoprotein) is largely responsible for host cell recognition and entry respectively. While the roles of the N-terminal residues of TM is well established as a fusion pore and anchor for Env into cell membranes, the role of the C-terminus of the protein is not well understood and is fiercely debated. This review gathers information on TM in an attempt to shed some light on the functional regions of this protein.
TL;DR: This study has determined the reliability of 10 housekeeping genes in context of four heavily studied viral infections; human immunodeficiency virus type 1, herpes simplex virustype 1, cytomegalovirus and varicella zoster virus infections using a variety of cell types and virus strains.
Abstract: The choice of an appropriate housekeeping gene for normalisation purposes has now become an essential requirement when designing QPCR experiments. This is of particular importance when using QPCR to measure viral and cellular gene transcription levels in the context of viral infections as viruses can significantly interfere with host cell pathways, the components of which traditional housekeeping genes often encode. In this study we have determined the reliability of 10 housekeeping genes in context of four heavily studied viral infections; human immunodeficiency virus type 1, herpes simplex virus type 1, cytomegalovirus and varicella zoster virus infections using a variety of cell types and virus strains. This provides researchers of these viruses with a shortlist of potential housekeeping genes to use as normalisers for QPCR experiments.
TL;DR: The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa, and results support a close homology between JP-A and JP-B and RsRNAV.
Abstract: RNA viruses have been isolated that infect marine organisms ranging from bacteria to whales, but little is known about the composition and population structure of the in situ marine RNA virus community. In a recent study, the majority of three genomes of previously unknown positive-sense single-stranded (ss) RNA viruses were assembled from reverse-transcribed whole-genome shotgun libraries. The present contribution comparatively analyzes these genomes with respect to representative viruses from established viral taxa. Two of the genomes (JP-A and JP-B), appear to be polycistronic viruses in the proposed order Picornavirales that fall into a well-supported clade of marine picorna-like viruses, the characterized members of which all infect marine protists. A temporal and geographic survey indicates that the JP genomes are persistent and widespread in British Columbia waters. The third genome, SOG, encodes a putative RNA-dependent RNA polymerase (RdRp) that is related to the RdRp of viruses in the family Tombusviridae, but the remaining SOG sequence has no significant similarity to any sequences in the NCBI database. The complete genomes of these viruses permitted analyses that resulted in a more comprehensive comparison of these pathogens with established taxa. For example, in concordance with phylogenies based on the RdRp, our results support a close homology between JP-A and JP-B and RsRNAV. In contrast, although classification of the SOG genome based on the RdRp places SOG within the Tombusviridae, SOG lacks a capsid and movement protein conserved within this family and SOG is thus likely more distantly related to the Tombusivridae than the RdRp phylogeney indicates.
TL;DR: By co-immunoprecipitation assay, a 74-KDa protein on the C6/36 cells binds JEV, and the mass spectrometry results indicated it may be heat shock cognate protein 70(HSC70) from Aedes aegypti.
Abstract: Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae. To date, the mechanisms of the early events of JEV infection remain poorly understood, and the cellular receptors are unidentified. There are evidences that the structure of the virus attachment proteins (VAP), envelope glycoprotein of mosquito-borne flaviviruses is very similar, and the vector-virus interaction of mosquito-borne flaviviruses is also very similar. Based on the studies previously demonstrated that the similar molecules present on the mosquito cells involved in the uptake process of JEV, West Nile virus (WNV) and Dengue virus (DV), it is proposed that the same receptor molecules for mosquito-borne flaviviruses (JEV, WNV and DV) may present on the surface of C6/36 mosquito cells. By co-immunoprecipitation assay, we investigated a 74-KDa protein on the C6/36 cells binds JEV, and the mass spectrometry results indicated it may be heat shock cognate protein 70(HSC70) from Aedes aegypti. Based upon some other viruses use of heat shock protein 70 (HSP70) family proteins as cell receptors, its possible HSC70's involvement in the fusion of the JEV E protein with the C6/36 cells membrane, and known form of cation channels in the interaction of HSC70 with the lipid bilayer, it will further be proposed that HSC70 as a penetration receptor mediates JEV entry into C6/36 cells.
TL;DR: This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.
Abstract: Background
The yellow fever virus, a member of the genus Flavivirus, is an arthropod-borne pathogen causing severe disease in humans. The attenuated yellow fever 17D virus strain has been used for human vaccination for 70 years and has several characteristics that are desirable for the development of new, live attenuated vaccines. We described here a methodology to construct a viable, and immunogenic recombinant yellow fever 17D virus expressing a green fluorescent protein variant (EGFP). This approach took into account the presence of functional motifs and amino acid sequence conservation flanking the E and NS1 intergenic region to duplicate and fuse them to the exogenous gene and thereby allow the correct processing of the viral polyprotein precursor.
TL;DR: Intranasal coadministration of M2e-MAP with infectious IAV merits further investigation in view of its potential applicability to human vaccination with live attenuated IAV and the role of adjuvants and route of immunization on Ab response and strength of protection.
Abstract: The M2 ectodomain (M2e) of influenza A virus (IAV) strains that have circulated in humans during the past 90 years shows remarkably little structural diversity. Since M2e-specific antibodies (Abs) are capable of restricting IAV replication in vivo but are present only at minimal concentration in human sera, efforts are being made to develop a M2e-specific vaccine. We are exploring a synthetic multiple antigenic peptide (MAP) vaccine and here report on the role of adjuvants (cholera toxin and immunostimulatory oligodeoxynucleotide) and route of immunization on Ab response and strength of protection. Independent of adjuvants and immunization route, on average 87% of the M2e-MAP-induced Abs were specific for M2e peptide and a variable fraction of these M2e(pep)-specific Abs (average 15%) cross-reacted with presumably native M2e expressed by M2-transfected cells. The titer of these cross-reactive M2e(pep-nat)-specific Abs in sera of parenterally immunized mice displayed a sigmoidal relation to level of protection, with EC50 of ~20 μg Ab/ml serum, though experiments with passive M2e(pep-nat) Abs indicated that serum Abs did not fully account for protection in parenterally vaccinated mice, particularly in upper airways. Intranasal vaccination engendered stronger protection and a higher proportion of G2a Abs than parenteral vaccination, and the strength of protection failed to correlate with M2e(pep-nat)-specific serum Ab titers, suggesting a role of airway-associated immunity in protection of intranasally vaccinated mice. Intranasal administration of M2e-MAP without adjuvant engendered no response but coadministration with infectious IAV slightly enhanced the M2e(pep-nat) Ab response and protection compared to vaccination with IAV or adjuvanted M2e-MAP alone. M2e-MAP is an effective immunogen as ~15% of the total M2e-MAP-induced Ab response is of desired specificity. While M2e(pep-nat)-specific serum Abs have an important role in restricting virus replication in trachea and lung, M2e-specific T cells and/or locally produced Abs contribute to protection in upper airways. Intranasal vaccination is preferable to parenteral vaccination, presumably because of induction of local protective immunity by the former route. Intranasal coadministration of M2e-MAP with infectious IAV merits further investigation in view of its potential applicability to human vaccination with live attenuated IAV.
TL;DR: The results of this study demonstrate that hydralazine and valproate can be safely administered to HPV-related malignancies such as cervical cancer because they do not increase viral oncoprotein expression.
Abstract: The methylation status at the human papilloma virus (HPV) genome found in pre-invasive and invasive cervical lesions suggests that neoplastic transformation can be suppressed by gene hypermethylation, whereas hypomethylation accompanies or causes cancer progression; hence, epigenetic therapy aimed at reactivating cellular suppressor-gene expression has the potential to act as a tumor promoter by enhancing HPV oncoprotein expression in HPV-related malignancies. The objective of this study was to determine the influence of hydralazine and valproate on HPV oncogene expression in cervical cancer cell lines and the primary tumors of patients undergoing treatment with hydralazine and valproate. Overall, hydralazine and valproate either alone or combined exerted a growth inhibitory effect on cervical cancer cell lines. A cell line-specific up-regulating effect was observed on E6/E7 gene expression, which in general correlated with DNA hypomethylation and histone acetylation at the long control region (LCR). Nonetheless, E6/E7 expression was unchanged or decreased in the majority of patients with cervical cancer treated with hydralazine, valproate, or both. In some cervical cancer cell lines, these drugs led to increased transcription of p53, and increased its stabilization due to acetylation at lysines 273 and 282, which allowed a higher bax-protein transactivating effect. The results of this study demonstrate that hydralazine and valproate can be safely administered to HPV-related malignancies such as cervical cancer because they do not increase viral oncoprotein expression. Most importantly, the antitumor effect of hydralazine and valproate in cervical cancer may at least partially depend on an up-regulating effect on p53 gene and on the valproate-induced hyperacetylation of p53 protein, protecting it from degradation by E6.
TL;DR: The present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.
Abstract: Chloroquine is a 4-aminoquinoline previously used in malaria therapy and now becoming an emerging investigational antiviral drug due to its broad spectrum of antiviral activities. To explore whether the low pH-dependency of influenza A viruses might affect the antiviral effects of chloroquine at clinically achievable concentrations, we tested the antiviral effects of this drug on selected human and avian viruses belonging to different subtypes and displaying different pH requirements. Results showed a correlation between the responses to chloroquine and NH4Cl, a lysosomotropic agent known to increase the pH of intracellular vesicles. Time-of-addition experiments showed that the inhibitory effect of chloroquine was maximal when the drug had been added at the time of infection and was lost after 2 h post-infection. This timing approximately corresponds to that of virus/cell fusion. Moreover, there was a clear correlation between the EC50 of chloroquine in vitro and the electrostatic potential of the HA subunit (HA2) mediating the virus/cell fusion process. Overall, the present study highlights the critical importance of a host cell factor such as intravesicular pH in determining the anti-influenza activity of chloroquine and other lysosomotropic agents.
TL;DR: The results indicate a markedly high prevalence of mixed A/D genotype infections in subjects with CAH and a possible association of mixed infections with the severity of liver diseases and the distribution of HBV A-D genotypes in pediatric cancer Egyptian patients.
Abstract: There are eight genotypes of hepatitis B virus (A-H) and subgenotypes are recognized. Genotyping can be accomplished based on a partial sequence of HBV genome such as the pre-S or S gene. Several methods have been developed and used for HBV genotyping. This study was undertaken to determine the HBV genotypes in Egyptian pediatric cancer patients with acute and chronic liver disease. HBV genotypes were determined in 22 patients who had acute forms of liver disease (AH) and in 48 patients with chronic active hepatitis (CAH). A type-specific primer based the nested-PCR method was employed in the HBV genotyping. This study showed that HBV infections in pediatric cancer patients are attributed predominantly to viral genotypes D and B that constituted 37.1% and 25.7%, respectively of the total infections. In addition, there was a relatively high prevalence of mixed infections of 15.7% among the studied group especially mixed A/D genotype infections. Genotype D was found significantly more often in patients with CAH than in patients with AH [23/48(47.9%) v 3/22 (13.6%)]. These findings show the distribution of HBV A-D genotypes in pediatric cancer Egyptian patients. Furthermore, our results indicate a markedly high prevalence of mixed A/D genotype infections in subjects with CAH and a possible association of mixed infections with the severity of liver diseases.
TL;DR: The study carried out to investigate the effects of inoculating three cowpea cultivars with three unrelated viruses showed that " OLOYIN" cultivar was most tolerant and produced reasonable yields when infected 30 DAP, while CABMV caused more severe effects in IT86D-719 and SBMV had the greatest effect on "OLO II".
Abstract: The study was carried out to investigate the effects of inoculating three cowpea cultivars: "OLO II", "OLOYIN" and IT86D-719 with three unrelated viruses: Cowpea aphid-borne mosaic virus (CABMV), genus Potyvirus, Cowpea mottle virus (CMeV), genus Carmovirus and Southern bean mosaic virus (SBMV), genus Sobemovirus singly and in mixture on growth and yield of cultivars at 10 and 30 days after planting (DAP). Generally, the growth and yield of the buffer inoculated control plants were significantly higher than those of the virus inoculated plants. Inoculation of plants at an early age of 10 DAP resulted in more severe effect than inoculations at a later stage of 30 DAP. The average values of plant height and number of leaves produced by plants inoculated 30 DAP were higher than those produced by plants inoculated 10 DAP. Most of the plants inoculated 10 DAP died and did not produce seeds. However, " OLOYIN" cultivar was most tolerant and produced reasonable yields when infected 30 DAP. The effect of single viruses on growth and yield of cultivars showed that CABMV caused more severe effects in IT86D-719, SBMV had the greatest effect on "OLO II" while CMeV induced the greatest effect on "OLOYIN". Yield was greatly reduced in double infections involving CABMV in combination with either CMeV or SBMV in "OLOYIN" and "OLO II", however, there was complete loss in yield of IT86D-719. Triple infection led to complete yield loss in all the three cultivars.
TL;DR: It is demonstrated that serially passaging liver homogenates from MARV-infected severe combined immunodeficient (scid) mice was highly successful in reducing the time to death in scid mice from 50–70 days to 7–10 days after MARV -Ci67, -Musoke, or -Ravn challenge.
Abstract: The filoviruses, Ebola (EBOV) and Marburg (MARV), cause a lethal hemorrhagic fever. Human isolates of MARV are not lethal to immmunocompetent adult mice and, to date, there are no reports of a mouse-adapted MARV model. Previously, a uniformly lethal EBOV-Zaire mouse-adapted virus was developed by performing 9 sequential passages in progressively older mice (suckling to adult). Evaluation of this model identified many similarities between infection in mice and nonhuman primates, including viral tropism for antigen-presenting cells, high viral titers in the spleen and liver, and an equivalent mean time to death. Existence of the EBOV mouse model has increased our understanding of host responses to filovirus infections and likely has accelerated the development of countermeasures, as it is one of the only hemorrhagic fever viruses that has multiple candidate vaccines and therapeutics. Here, we demonstrate that serially passaging liver homogenates from MARV-infected severe combined immunodeficient (scid) mice was highly successful in reducing the time to death in scid mice from 50–70 days to 7–10 days after MARV-Ci67, -Musoke, or -Ravn challenge. We performed serial sampling studies to characterize the pathology of these scid mouse-adapted MARV strains. These scid mouse-adapted MARV models appear to have many similar properties as the MARV models previously developed in guinea pigs and nonhuman primates. Also, as shown here, the scid-adapted MARV mouse models can be used to evaluate the efficacy of candidate antiviral therapeutic molecules, such as phosphorodiamidate morpholino oligomers or antibodies.