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.     

The Mode of Inhibition of TMV- and PVX-Induced RNA-Dependent RNA Polymerases by some Antiphytoviral Drugs

Six nucleobase and nucleoside analogues (6-aminouracil [6-AU], 6-aminothymin [6-AT], 6-fluorothymin [6-FT], 2,4-dioxohexahydro-1,3,5-triazine [DHT], 9-(2,3-dihydroxypropyl) adenine [DHPA] and 1-β-D-ribofuranosyl-1,2,4-triazol-3-carboxamid [ribavirin]) with potential antiphytoviral activity were tested in an in vitro replication system for their effect on tobacco mosaic virus (TMV)-and potato virus X(PVX)-induced RNA dependent RNA polymerase. 6-AU, 6-AT, 6-FT, and DHPA inhibited both polymerases which may be a target of these compounds. A Putative noncompetitive mode of inhibition is discussed. Whereas ribavirin did not affect the in vitr RNA synthesis in the established systems, the uracil-derivative DHT inhibited only the PVX-specific enzyme.     

The cassava mosaic geminiviruses occurring in Uganda following the 1990s epidemic of severe cassava mosaic disease

The cassava mosaic geminiviruses (CMGs) isolated from cassava plants expressing mild and severe symptoms of cassava mosaic disease (CMD) in 2002 in Uganda were investigated using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) molecular techniques and DNA sequencing. Two previously described cassava mosaic geminiviruses: African cassava mosaic virus (ACMV) said East African cassava mosaic virus - Uganda variant (EACMV-UG2) were detected in Uganda. The RFLP technique distinguished two polymorphic variants of ACMV (ACMV-UG1 and ACMV-UG2) and three of EACMV-UG2 (EACMV-UG2[1], EACMV-UG2[2] and EACMV-UG2[3]). ACMV-UG1 produced the fragments predicted for the published sequences of ACMV-[KE]/UGMld/ UGSvr, while ACMV-UG2, which produced the RFLP fragments predicted for the West African ACMV isolates ACMV-[NG], ACMV-[CM], ACMV-[CM/DO2] and ACMV-[CI], was shown to be ACMV-UGMld/UGSvr after DNA sequencing. EACMV-UG2[1] produced the RFLP fragments predicted for the published sequences of EACMV-UG2/UG2Mld/UG2Svr. However, both EACMV-UG2[2] and EACMV-UG2[3], which produced East African cassava mosaic vzras-[Tanzania]-like polymorphic fragments with RFLP analysis, were confirmed to be isolates of EACMV-UG2 after DNA sequencing. Thus, this study emphasises the importance of DNA sequence analysis for the identification of CMG isolates. EACMV-UG2 was the predominant virus and occurred in all the surveyed regions. It was detected in 73% of the severely and 53% of the mildly diseased plants, while ACMV was less widespread and occurred most frequently in the mildly diseased plants (in 27% of these plants). Mixed infections of ACMV and EACMV-UG2 were detected in only 18% of the field samples. Unlike previously reported results the mixed infection occurred almost equally in plants exhibiting mild or severe disease symptoms (21% and 16%, respectively). The increasing frequency of mild forms of EACMV-UG2 together with the continued occurrence of severe forms in the field warrants further studies of virus-virus and virus-host interactions.     

Generation of multivirus-specific T cells by a single stimulation of peripheral blood mononuclear cells with a peptide mixture using serum-free medium

Background: Restoration of virus-specific immunity by virus specific T cells (VSTs) offers an attractive alternative to conventional drugs, and can be highly effective in immunocompromised patients, including hematopoietic stem cell transplant (HSCT) recipients. However, conventional VSTs manufacture requires preparation of specialized antigen-presenting cells (APCs), prolonged ex vivo culture in serum-containing medium and antigen re-stimulation with viruses or viral vectors to provide viral antigens for presentation on APCs. Methods: To simplify this complex process, we developed a method to generate multiple VSTs by direct stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries in serum-free medium. Results: We generated VSTs that targeted seven viruses (cytomegalovirus [CMV], Epstein-Barr virus [EBV], adenovirus [AdV], human herpesvirus 6 [HHV-6], BK virus [BKV], JC virus [JCV] and Varicella Zoster virus [VZV]) in a single line. The phenotype, growth and specificity of multiple VSTs produced in serum-free medium were equivalent to those generated in conventional serum-containing medium. Discussion: The use of serum-free medium allows this approach to be readily introduced to clinical practice with lower cost, greater reproducibility due to the absence of batch-to-batch variability in serum and without concerns for infectious agents in the serum used. This simplified approach will now be tested in recipients of Human Leukocyte Antigen (HLA)–matched sibling HSCT.     

Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope

Aichi virus (AiV) is an emerging single-stranded, positive-sense, non-enveloped RNA virus in the Picornaviridae that causes acute gastroenteritis in humans. The first case of AiV infection in Taiwan was diagnosed in a human neonate with enterovirus-associated symptoms; the virus was successfully isolated and propagated. To establish a method to detect AiV, we analyzed the antigen epitope and generated a polyclonal antibody against AiV viral protein 1 (VP1). This peptide-purified anti-AiV VP1 antibody showed high specificity against AiV VP1 without cross-reaction to nine other tested strains of Picornaviruses. The anti-AiV VP1 antibody was used in immunofluorescence analysis, immunoblotting, and enzyme-linked immunosorbent assay to elucidate the cell tropism and replication kinetics of AiV. Use of the anti-AiV VP1 antibody also revealed AiV infection restriction with interferon type I and polyI/C antiviral treatment. The AiV infection and detection system may provide an in vitro platform for AiV virology study.     

Antiviral RNA recognition and assembly by RLR family innate immune sensors

Virus-encoded molecular signatures, such as cytosolic double-stranded or otherwise biochemically distinct RNA species, trigger cellular antiviral signaling. Cytoplasmic proteins recognize these non-self RNAs and activate signal transduction pathways that drive the expression of virus-induced genes, including the primary antiviral cytokine, IFNβ, and diverse direct and indirect antiviral effectors [1], [2], [3], [4]. One important group of cytosolic RNA sensors known as the RIG-I-like receptors (RLRs) is comprised of three proteins that are similar in structure and function. The RLR proteins, RIG-I, MDA5, and LGP2, share the ability to recognize nucleic acid signatures produced by virus infections and activate antiviral signaling. Emerging evidence indicates that RNA detection by RLRs culminates in the assembly of dynamic multimeric ribonucleoprotein (RNP) complexes. These RNPs can act as signaling platforms that are capable of propagating and amplifying antiviral signaling responses. Despite their common domain structures and similar abilities to induce antiviral responses, the RLRs differ in their enzymatic properties, their intrinsic abilities to recognize RNA, and their ability to assemble into filamentous complexes. This molecular specialization has enabled the RLRs to recognize and respond to diverse virus infections, and to mediate both unique and overlapping functions in immune regulation [5], [6].     

Characterization of the envelope proteins of pseudorabies virus.

Previously we have reported that among the proteins of purified pseudorabies virions there are four major glycoproteins (T. Ben-Porat and A. S. Kaplan, Virology 41:265-273, 1970). Several minor glycoproteins can also be identified by two-dimensional gel electrophoresis. Removal of the viral envelope with Triton X-100 selectively removes from the virions all of the glycoproteins as well as several non-glycosylated proteins. Sedimentation analysis or chromatography of these proteins reveals that several are complexed with one another, some being covalently linked via disulfide bridges. Analysis of the proteins by immunoprecipitation with monoclonal antibodies reactive with the membrane proteins showed also that three of the four major virus glycoproteins (125K, 74K, and 58K; gIIa, gIIb, and gIIc, respectively) are linked covalently by disulfide bridges. Furthermore, all three share extensive sequence homology as indicated by the identity of their antigenic determinants and by partial peptide mapping; they probably originate from a single protein precursor. The fourth major glycoprotein (98K; gIII) is not complexed to any other protein. Three minor glycoproteins (130K [gI], 98K [gIV], and 62K [gV]), which form a noncovalently linked complex with a 115K nonglycosylated protein, have also been identified. Of the monoclonal antibodies used in this study, only those reactive with the major 98K glycoprotein (gIII) inhibit virus adsorption and neutralize virus infectivity in the absence of complement. However, all react with surface components of the virion, indicating that the proteins with which they react are exposed on the surface of the virions. A nomenclature for the pseudorabies virus glycoproteins is proposed.     

Isolation and characterization of a single-stranded RNA virus infecting the marine planktonic diatom Chaetoceros tenuissimus Meunier

Diatoms are important components of the biological community and food web in the aquatic environment. Here, we report the characteristics of a single-stranded RNA (ssRNA) virus (CtenRNAV01) that infects the marine diatom Chaetoceros tenuissimus Meunier (Bacillariophyceae). The ca. 31-nm virus particle is icosahedral and lacks a tail. CtenRNAV01 forms crystalline arrays occupying most of the infected host's cytoplasm. By growth experiments, the lytic cycle and the burst size were estimated to be <24 h and approximately 1 x 10(4) infectious units per host cell, respectively. Stationary-phase C. tenuissimus cultures were shown to be more sensitive to CtenRNAV01 than logarithmic-phase cultures. The most noticeable feature of this virus is its exceptionally high yields of approximately 10(10) infectious units ml(-1); this is much higher than those of any other algal viruses previously characterized. CtenRNAV01 has two molecules of ssRNA of approximately 8.9 and 4.3 kb and three major proteins (33.5, 31.5, and 30.0 kDa). Sequencing of the total viral genome has produced only one large contig [9,431 bases excluding the poly(A) tail], suggesting considerable overlapping between the two RNA molecules. The monophyly of CtenRNAV01 compared to another diatom-infecting virus, Rhizosolenia setigera RNA virus, was strongly supported in a maximum likelihood phylogenetic tree constructed based on the concatenated amino acid sequences of the RNA-dependent RNA polymerase domains. Although further analysis is required to determine the detailed classification and nomenclature of this virus, these data strongly suggest the existence of a diatom-infecting ssRNA virus group in natural waters.     

Arrangement of ceramide [EOS] in a stratum corneum lipid model matrix: new aspects revealed by neutron diffraction studies

The lipid matrix in stratum corneum (SC) plays a key role in the barrier function of the mammalian skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). Especially the unique-structured omega-acylceramide CER[EOS] is regarded to be essential for skin barrier properties by inducing the formation of a long-periodicity phase of 130 angstroms (LPP). In the present study, the arrangement of CER[EOS], either mixed with CER[AP] and CHOL or with CER[AP], CHOL and palmitic acid (PA), inside a SC lipid model membrane has been studied for the first time by neutron diffraction. For a mixed CER[EOS]/CER[AP]/CHOL membrane in a partly dehydrated state, the internal membrane nanostructure, i.e. the neutron scattering length density profile in the direction normal to the surface, was obtained by Fourier synthesis from the experimental diffraction patterns. The membrane repeat distance is equal to that of the formerly used SC lipid model system composed of CER[AP]/CHOL/PA/ChS. By comparing both the neutron scattering length density profiles, a possible arrangement of synthetic long-chain CER[EOS] molecules inside a SC lipid model matrix is suggested. The analysis of the internal membrane nanostructure implies that one CER[EOS] molecule penetrates from one membrane layer into an adjacent layer. A 130 angstroms periodicity phase could not be observed under experimental conditions, either in CER/CHOL mixtures or in CER/CHOL/FFA mixture. CER[EOS] can be arranged inside a phase with a repeat unit of 45.2 angstroms which is predominately formed by short-chain CER[AP] with distinct polarity.     

A mutant killer plasmid whose replication depends on a chromosomal "superkiller" mutation

Yeast strains carrying a 1.5 x 10(6) molecular weight linear double-stranded RNA in virus-like particles (M dsRNA, the killer plasmid or virus) secrete a toxin that is lethal to strains not carrying this plasmid. Recessive mutations in any of four chromosomal genes (called ski1-ski4) result in increased production of toxin activity. We report here a mutation of the killer plasmid (called [KIL-sd] for ski-dependent) that makes the killer plasmid dependent for its replication on the presence of a chromosomal mutation in any ski gene. Thus, the [KIL-sd] plasmid is lost from SKI(+) strains. When the wild-type killer plasmid, [KIL-k], is introduced into a ski2-2 [KIL-o] strain, the killer plasmid changes to a [KIL-sd] plasmid. This may represent a specific form of mutagenesis or selective replication in the ski2-2 strain of [KIL-sd] variants (mutants) in the normal [KIL-k] population. The ski2-1 and ski2-3 mutations do not convert [KIL-k] to [KIL-sd], but ski2-3 does allow maintenance of the [KIL-sd] plasmid. The [KIL-sd] plasmid thus lacks a plasmid site or product needed for replication in wild-type cells.     

RNA病毒感染性克隆技术及其应用

为实现对RNA病毒基因分子水平上的操作与研究,可将病毒基因组RNA体外转换成cDNA,构建出cDNA克隆,通过cDNA克隆与重组技术实现对RNA病毒基因分子水平的修饰,从而为病毒基因组的结构和功能研究提供有效的方法。这种方法是现代实验病毒学非常有用的工具,称为RNA病毒感染性克隆技术或反向遗传学技术。我们对该技术及其应用进行简要综述。     

Summary of the 9th annual meeting of the italian society for virology

The 9th annual meeting of the Italian Society for Virology (SIV) comprised seven plenary sessions focused on: General virology and viral genetics; Virus–Host interaction and pathogenesis; Viral oncology; Emerging viruses and zoonotic, foodborne, and environmental pathways of transmission; Viral immunology and vaccines; Medical virology and antiviral therapy; Viral biotechnologies and gene therapy. Moreover, four hot topics were discussed in special lectures: the Pioneer in human virology lecture regarding the control of viral epidemics with particular emphasis on the human immunodeficiency virus (HIV), the Pioneer in plant virology lecture focused on cell responses to plant virus infection, a Keynote lecture on the epidemiology and genetic diversity of Crimea–Congo Hemorrhagic Fever virus, and the G.B. Rossi lecture on the molecular basis and clinical implications of human cytomegalovirus tropism for endothelial/epithelial cells. The meeting had an attendance of about 160 virologists. A summary of the plenary lectures and oral selected presentations is reported. J. Cell. Physiol. 226: 285–287, 2010. © 2010 Wiley‐Liss, Inc.     

Sequence heterogeneity in NS5A of hepatitis C virus genotypes 2a and 2b and clinical outcome of pegylated-interferon/ribavirin therapy

Pegylated-interferon plus ribavirin (PEG-IFN/RBV) therapy is a current standard treatment for chronic hepatitis C. We previously reported that the viral sequence heterogeneity of part of NS5A, referred to as the IFN/RBV resistance-determining region (IRRDR), and a mutation at position 70 of the core protein of hepatitis C virus genotype 1b (HCV-1b) are significantly correlated with the outcome of PEG-IFN/RBV treatment. Here, we aimed to investigate the impact of viral genetic variations within the NS5A and core regions of other genotypes, HCV-2a and HCV-2b, on PEG-IFN/RBV treatment outcome. Pretreatment sequences of NS5A and core regions were analyzed in 112 patients infected with HCV-2a or HCV-2b, who were treated with PEG-IFN/RBV for 24 weeks and followed up for another 24 weeks. The results demonstrated that HCV-2a isolates with 4 or more mutations in IRRDR (IRRDR[2a]≥4) was significantly associated with rapid virological response at week 4 (RVR) and sustained virological response (SVR). Also, another region of NS5A that corresponds to part of the IFN sensitivity-determining region (ISDR) plus its carboxy-flanking region, which we referred to as ISDR/+C[2a], was significantly associated with SVR in patients infected with HCV-2a. Multivariate analysis revealed that IRRDR[2a]≥4 was the only independent predictive factor for SVR. As for HCV-2b infection, an N-terminal half of IRRDR having two or more mutations (IRRDR[2b]/N≥2) was significantly associated with RVR, but not with SVR. No significant correlation was observed between core protein polymorphism and PEG-IFN/RBV treatment outcome in HCV-2a or HCV-2b infection. CONCLUSION: The present results suggest that sequence heterogeneity of NS5A of HCV-2a (IRRDR[2a]≥4 and ISDR/+C[2a]), and that of HCV-2b (IRRDR[2b]/N≥2) to a lesser extent, is involved in determining the viral sensitivity to PEG-IFN/RBV therapy.     

Rescue of infectious rift valley fever virus entirely from cDNA, analysis of virus lacking the NSs gene, and expression of a foreign gene

Rift Valley fever virus (RVFV) (genus Phlebovirus, family Bunyaviridae) has a tripartite negative-strand genome, causes a mosquito-borne disease that is endemic in sub-Saharan African countries and that also causes large epidemics among humans and livestock. Furthermore, it is a bioterrorist threat and poses a risk for introduction to other areas. In spite of its danger, neither veterinary nor human vaccines are available. We established a T7 RNA polymerase-driven reverse genetics system to rescue infectious clones of RVFV MP-12 strain entirely from cDNA, the first for any phlebovirus. Expression of viral structural proteins from the protein expression plasmids was not required for virus rescue, whereas NSs protein expression abolished virus rescue. Mutants of MP-12 partially or completely lacking the NSs open reading frame were viable. These NSs deletion mutants replicated efficiently in Vero and 293 cells, but not in MRC-5 cells. In the latter cell line, accumulation of beta interferon mRNA occurred after infection by these NSs deletion mutants, but not after infection by MP-12. The NSs deletion mutants formed larger plaques than MP-12 did in Vero E6 cells and failed to shut off host protein synthesis in Vero cells. An MP-12 mutant carrying a luciferase gene in place of the NSs gene replicated as efficiently as MP-12 did, produced enzymatically active luciferase during replication, and stably retained the luciferase gene after 10 virus passages, representing the first demonstration of foreign gene expression in any bunyavirus. This reverse genetics system can be used to study the molecular virology of RVFV, assess current vaccine candidates, produce new vaccines, and incorporate marker genes into animal vaccines.     

Not Beyond Reasonable Doubt: Howard Temin’s Provirus Hypothesis Revisited

During the 1960s, Howard M. Temin (1934–1994), dared to advocate a “heretical” hypothesis that appeared to be at variance with the central dogma of molecular biology, understood by many to imply that information transfer in nature occurred only from DNA to RNA. Temin’s provirus hypothesis offered a simple explanation of both virus replication and viral-induced cancer and stated that Rous sarcoma virus, an RNA virus, is replicated via a DNA intermediate. Popular accounts of this scientific episode, written after the discovery of an RNA-directed DNA polymerase in 1970, tend to describe the reaction to his proposition as ardent opposition. Typically these accounts use a ‹molecular biology’ standpoint emphasizing the central dogma’s part in its rejection. In this article, however, this episode will be examined from a joint perspective of virology and experimental cancer research. From this perspective it is clear that Temin’s work was well within the epistemological and methodological boundaries of virology and cancer research. Still, scientists did have␣reasons to doubt the provirus hypothesis, but these do not seem to be good enough to either justify an account that portrays Temin as a renegade or his ideas as heretical.     

Replication and packaging of Turnip yellow mosaic virus RNA containing Flock house virus RNA1 sequence

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA as a genome. In this study, RNA1 sequence of Flock house virus (FHV) was inserted into the TYMV genome to test whether TYMV can accommodate and express another viral entity. In the resulting construct, designated TY-FHV, the FHV RNA1 sequence was expressed as a TYMV subgenomic RNA. Northern analysis of the Nicotiana benthamiana leaves agroinfiltrated with the TY-FHV showed that both genomic and subgenomic FHV RNAs were abundantly produced. This indicates that the FHV RNA1 sequence was correctly expressed and translated to produce a functional FHV replicase. Although these FHV RNAs were not encapsidated, the FHV RNA having a TYMV CP sequence at the 3’-end was efficiently encapsidated. When an eGFP gene was inserted into the B2 ORF of the FHV sequence, a fusion protein of B2-eGFP was produced as expected. [BMB Reports 2014; 47(6): 330-335]