Relevant Viruses Research Articles

RNAi-mediated immunity provides strong protection against the negative-strand RNA vesicular stomatitis virus in Drosophila

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.

Analytical Methods for Virus Detection in Water and Food

Potential ways to address the issues that relate to the techniques for analyzing food and environmental samples for the presence of enteric viruses are discussed. It is not the authors’ remit to produce or recommend standard or reference methods but to address specific issues in the analytical procedures. Foods of primary importance are bivalve molluscs, particularly, oysters, clams, and mussels; salad crops such as lettuce, green onions and other greens; and soft fruits such as raspberries and strawberries. All types of water, not only drinking water but also recreational water (fresh, marine, and swimming pool), river water (irrigation water), raw and treated sewage are potential vehicles for virus transmission. Well over 100 different enteric viruses could be food or water contaminants; however, with few exceptions, most well-characterized foodborne or waterborne viral outbreaks are restricted to hepatitis A virus (HAV) and calicivirus, essentially norovirus (NoV). Target viruses for analytical methods include, in addition to NoV and HAV, hepatitis E virus (HEV), enteroviruses (e.g., poliovirus), adenovirus, rotavirus, astrovirus, and any other relevant virus likely to be transmitted by food or water. A survey of the currently available methods for detection of viruses in food and environmental matrices was conducted, gathering information on protocols for extraction of viruses from various matrices and on the various specific detection techniques for each virus type.

Single and Multiple Viral Infections in Lower Respiratory Tract Infection

Background: Lower respiratory tract infection (LRTI) often leads to hospitalization, and it was indicated that causative viral infections are underestimated. Objectives: It was our aim to compare the frequency of 8 relevant viruses in 109 hospitalized LRTI patients and 144 healthy controls. Methods: Virus infection was determined by seroconversion and ELISA for anti-virus antibodies in repeated serum samples. Bacterial infection was diagnosed in respiratory specimens, blood cultures and urine. Results: The LRTI patient cohort consisted of 49 patients with community-acquired pneumonia, 30 patients with acute bronchitis and 30 chronic obstructive pulmonary disease patients with acute exacerbation. Viral infection was detected in 89 (82%) LRTI patients compared with 32 (22%) in healthy controls (relative risk 3.42, 95% confidence interval 2.48–4.72; p < 0.0001). The most frequent viral pathogens were: influenza B (23%), adenovirus (16%) and parainfluenza virus 3 (12%). Importantly, infections with more than 1 virus were detected in 63% (n = 57) of LRTI patients with viral infection, which represents 52% of all LRTI patients. No multiple virus infection was detected in the healthy controls. Patients with community-acquired pneumonia were more often infected with adenovirus and respiratory syncytial virus as compared with the other LRTI patients (p = 0.046 and 0.0009, respectively). Conclusions: There is a high incidence of single and multiple viral infections in LRTI patients requiring hospitalization. The data indicate the need for regular virus diagnosis and the development of point of care tools that enables a fast diagnosis of the most common viruses and bacteria. The data also imply the need to consider antiviral therapy in positive LRTI cases.

Protein Analysis of Purified Respiratory Syncytial Virus Particles Reveals an Important Role for Heat Shock Protein 90 in Virus Particle Assembly

In this study, we used imaging and proteomics to identify the presence of virus-associated cellular proteins that may play a role in respiratory syncytial virus (RSV) maturation. Fluorescence microscopy of virus-infected cells revealed the presence of virus-induced cytoplasmic inclusion bodies and mature virus particles, the latter appearing as virus filaments. In situ electron tomography suggested that the virus filaments were complex structures that were able to package multiple copies of the virus genome. The virus particles were purified, and the protein content was analyzed by one-dimensional nano-LC MS/MS. In addition to all the major virus structural proteins, 25 cellular proteins were also detected, including proteins associated with the cortical actin network, energy pathways, and heat shock proteins (HSP70, HSC70, and HSP90). Representative actin-associated proteins, HSC70, and HSP90 were selected for further biological validation. The presence of beta-actin, filamin-1, cofilin-1, HSC70, and HSP90 in the virus preparation was confirmed by immunoblotting using relevant antibodies. Immunofluorescence microscopy of infected cells stained with antibodies against relevant virus and cellular proteins confirmed the presence of these cellular proteins in the virus filaments and inclusion bodies. The relevance of HSP90 to virus infection was examined using the specific inhibitors 17-N-Allylamino-17-demethoxygeldanamycin. Although virus protein expression was largely unaffected by these drugs, we noted that the formation of virus particles was inhibited, and virus transmission was impaired, suggesting an important role for HSP90 in virus maturation. This study highlights the utility of proteomics in facilitating both our understanding of the role that cellular proteins play during RSV maturation and, by extrapolation, the identification of new potential targets for antiviral therapy.

Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed.

Hot Topic: [Herpesvirus Vectors in Gene Therapy (Guest Editor: Veijo Hukkanen)

Herpes simplex virus type 1 (HSV-1) is a common human virus, best known as the causative agent of recurrent labial herpes (cold sores) [1]. The unique properties of HSV-1 have increasingly been exploited in vector design, particularly for therapy of the central nervous system diseases. HSV has natural properties useful for a therapy vector: the latent, life-long infections in the nervous system with long-term gene expression, the episomal non-integrating DNA genome and the numerous dispensable genes allowing cloning of large transgenes and their regulatory sequences [2]. Numerous clinical phase I-III studies using HSV vectors are in progress on therapy of gliomas and other malignancies and of other, mainly neurological, disorders, including pain [3, 4]. The HSV vectors can be genomic, nonreplicating or replicative vectors, or HSV amplicons. HSV amplicons are vectors harboring only minimal amount of virally derived sequences, suitable for delivery of up to 150 kbp of foreign DNA [5]. The use of HSV amplicon vectors has further been improved by the development of HSV-adeno-associated virus (AAV) hybrid vectors, aiming at long-term transgene expression. The wild type HSV, and also the HSV vectors, can evoke both innate and adaptive immune responses in the host organism [6]. The host responses can limit the vector persistence and duration of the transgene expression, but may occasionally also be advantageous in therapy of certain diseases. The current special issue introduces to the reader the different types and uses of HSV vectors, as well as the host responses which may affect the performance of these vectors. In this issue, Tsitoura and Epstein provide an expert overview of the HSV amplicon vectors and the host responses. They discuss the challenges in overcoming the amplicon silencing in the host, as well as the roles of certain HSV genes in counteracting the host responses. The review by de Oliveira and Fraefel introduces the HSV/AAV hybrid vectors, and illustrates also the biology of AAV and AAV vectors. The relevant helper viruses and HSV vectors are discussed, and new hybrid virus vectors are presented, such as HSV/Epstein-Barr virus and HSV/retrovirus hybrids. The overview by Cassady and Parker elucidates the use of HSV in tumor killing. They present the conditionally replicating HSV vectors for brain tumor therapy. In their paper, various approaches are illustrated for enhancement of tumor killing by HSV, such as combination of HSV virotherapy with other therapeutic modalities, or the tumor-specific targeting of HSV vectors. The authors discuss strategies to facilitate replication of HSV vectors in malignant cells without restoration of neurovirulence. In an extensive review, Manservigi et al. elucidate the design and applications of recombinant HSV vectors, with an emphasis on the genomic HSV vectors. They present the key issues of the HSV life cycle relevant to the vector development and introduce the different types of HSV vectors. The applications of HSV vectors are described in therapy of chronic diseases of the nervous system, in cancer therapy and also in vaccine vector design. The targeting strategies and means of imaging of HSV vectors are illustrated, and the authors also discuss the host responses and other factors compromizing the HSV vector efficacy. New perspectives for HSV-mediated tumor therapy are discussed, including the exploitation of microRNAs. This special issue of The Open Virology Journal presents state-of-the-art reviews by experts of the HSV vector field, covering the different types and uses of HSV vectors and elucidating the central questions such as vector design and targeting, silencing of the transgenes and other effects of the host responses. Hopefully this special issue is useful for the readership; for those interested in applications of HSV and for those considering the choice of viral vector for their particular gene therapy purpose.

Developing Vaccines to Combat Pandemic Influenza

Influenza vaccine manufacturers require antigenically relevant vaccine viruses that have good manufacturing properties and are safe to use. In developing pandemic vaccine viruses, reverse genetics has been employed as a rational approach that can also be used effectively to attenuate the highly virulent H5N1 virus and at the same time place the H5 HA and N1 NA on a background of PR8, a virus that has been used over many decades to provide high yielding vaccine viruses. Reverse genetics has also been used successfully alongside classical reassorting techniques in the development of (swine flu) pandemic A(H1N1)v vaccine viruses.

Response: Improving Development of the Molecular Signature for Diagnosis of Acute Respiratory Viral Infections

We are most pleased Statnikov et al. (2010)xStatnikov, A., McVoy, L., Lytkin, N., and Aliferis, C.F. Cell Host Microbe. 2010; 7: 100–101Abstract | Full Text | Full Text PDF | PubMed | Scopus (5)See all ReferencesStatnikov et al. (2010) could utilize the gene-expression data from Zaas et al. (Zaas et al., 2009xZaas, A., Chen, M., Varkey, J., Veldman, T., Hero, A.O. III, Lucas, J., Huang, Y., Turner, R., Gilbert, A., Lambkin-Williams, R. et al. Cell Host Microbe. 2009; 6: 207–217Abstract | Full Text | Full Text PDF | PubMed | Scopus (138)See all ReferencesZaas et al., 2009) to develop a similar gene-expression signature for viral infection. It is not surprising that microarray data can result in predictors that are not unique and that can offer signatures of comparable predictive performance. Ein-Dor et al. (2005)xEin-Dor, L., Kela, I., Getz, G., Givol, D., and Domany, E. Bioinformatics. 2005; 21: 171–178Crossref | PubMed | Scopus (481)See all ReferencesEin-Dor et al. (2005) identified at least eight separate gene sets whose expression patterns stratify the same patient into comparable risk categories for breast cancer suggesting that there are multiple forms of information in a single data set that can equally well stratify patients. The fundamental reason is the diversity, scale, and complexity of genomic factors defining the disease state.We appreciate Stanlikov et al.'s suggestions for improving the analysis and respond below in order of the suggestions. First, genes were selected based on coherent expression across the entire data set. Fitting the factor model was done without regard to phenotype. The appropriate factor from which to select genes was clear regardless of the samples used in cross-validation. Genes were selected and the model was built on the training data set only. Second, it is possible that the gene-expression patterns at baseline are predictive of symptomatic versus asymptomatic at the time of maximum symptoms (T). If this were the case, however, the effect would work against our ability to generate a model to distinguish between symptomatic and asymptomatic or the baseline healthy state. Thus, we are able to distinguish symptomatic from asymptomatic/baseline despite rather than because of any effect linking samples from baseline and T. Third, our analysis was purely unsupervised—no use of the phenotype data was made in the process of fitting the factor models. Part of the hierarchical structure of the Bayesian model includes the estimation of factor size, which is the proper technique for accounting for false discovery in this context. The choice of 30 genes was arbitrary but this choice does not in any way lessen the value of the result. Our goal was to present a gene set in the factors that might be amenable to a clinical assay such as RT PCR. Fourth, Statnikov et al. are correct that only one of the three viruses were in Ramilo et al. (2007)xRamilo, O., Allman, W., Chung, W., Mejias, A., Ardura, M., Glaser, C., Wittkowski, K.M., Piqueras, B., Banchereau, J., Palucka, A.K. et al. Blood. 2007; 109: 2066–2077Crossref | PubMed | Scopus (223)See all ReferencesRamilo et al. (2007). However, the goal of this aspect of our analysis was to show that the “acute respiratory viral signature” could identify one of the relevant viruses in the training set. Moreover, the Ramilo data set was generated on an independent microarray platform from that of ours. The fact that we could achieve such a good validation speaks to the robust nature of our acute respiratory signature. It should also be noted that we did treat each of the viral data sets as validations of the other (closer to “apples to apples”), and we are not aware of the existence of any public data set that is more appropriate for validation than Ramilo. Fifth, our work was not designed to demonstrate a complete analysis of the data, but rather to demonstrate the possibility of distinguishing infectious agents based on host response.

The role of viruses in oral disease

The focus has traditionally been on bacteria and fungi when discussing microbiological aspects of oral disease. Viruses are probably more involved in diseases associated with the oral cavity than has been previously thought. The role of several viruses in ulceration is well known, but viruses of the herpes family may play a role in periodontitis, and papillomaviruses are probably involved in oral cancer. This review offers a brief introduction to virology before discussing the role of the more relevant viruses in oral disease. As to clinical application, it is concluded that the anti-herpes medication may, in some cases, be relevant in treating periodontitis, while papillomavirus vaccine would be expected to decrease the prevalence of oral cancer.

Prediction and Comparative Analysis of MHC Binding Peptides and Epitopes in Nanoviridae Nano-organisms

Nanoviridae is a family of single stranded DNA viruses which infect the plants through their phloem tissues. The few members of these viruses are now turning towards animals. It includes two genera, Nanovirus and Babuvirus. Nanovirus includes three species namely Faba beans necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MVDV) and Subterranean clover stunt virus (SCSV) while Babuvirus have two species accounted yet, namely Abaca bunchy top virus (ABTV) and Banana bunchy top virus (BBTV). The viral coat proteins are likely to be responsible for many diseases in plants as well as animals. The intra-genomic changes or post translational modifications with in SCSV have decreased the length of the sequence, but increased the antigenic potential and numbers of antigenic binding regions on the surface of the protein. We have predicted the most probable responsible antigenic determinants and MHC binders within the viruses in Nanoviridae family. MHC molecules play a crucial role in host immune response. The nonamers of antigenic determinants in Nanoviridae family viral coat proteins are highly sensitive to H-2Kd and I-Ag7 molecules. The species within particular genera shows their common epitopes along with diverse colleague epitopes. These common antigenic peptides can be used as their identifi ers. These identifi ers along with their diverse colleagues could be most informative for antidotes production against themselves. Also, these are important for synthetic peptide vaccine production against the relevant viruses.

Quality and Safety in Blood Supply in 2010

The past two decades saw tremendous achievements in blood safety, which are due to the commitment of blood establishments and industry, progress in technology such as the improvement of serological and NAT screening tests, and stringent regulatory control. Milestones in the legislation were the inclusion of plasma derivatives in the pharmaceutical legislation of the European Community (EC) in the year 1989 and special laws for the blood sector in EC and in member states, such as the Transfusionsgesetz (Transfusion Law) in Germany. The legal frame has to be supplemented by scientific and technical guidance, which is provided on the European level by the European Directorate for the Quality of Medicines and Health Care and by the European Medicines Agency. In the member states, guidelines taking into account the national peculiarities can be elaborated, such as the German hemotherapy guidelines issued by the German Medical Association (BundesÄrztekammer) in agreement with the Paul-Ehrlich-Institut. The regulatory control of screening tests, and the introduction of NAT testing lead to a remarkably high degree of safety concerning the most relevant viruses HIV, HBV and HCV. Issues needing further attention are bacterial contamination and transfusion-associated acute lung injury (TRALI). Measures aiming at minimizing risks have to be balanced against their impact on supply. In order to ensure the assured supply with safe blood products, sustained efforts and research are needed as well as a continuous dialogue among blood services, industry, physicians, patients and regulatory authorities.

Quarantine Plasma: Quo vadis?

Upon the introduction of mandatory nucleic acid amplification technology (NAT) testing in Germany for HCV, quarantining of fresh frozen plasma (FFP) was reduced in 2002 from 6 to 4 months. In 2004 HIV-1 NAT and in 2005 anti-HBc testing were introduced to further reduce the residual transmission risks for transfusion relevant viruses. After testing more than 40 million donations by HCV NAT it became obvious that NAT testing has a very significant impact on viral blood safety by reducing the residual risk by a factor of 10. Only one documented HCV transmission occurred during more than 10 years of NAT testing in Germany, indicating that the remaining risk is marginal. Similar data were obtained for HIV-1. The question arises whether we could discontinue quarantining of FFP or further reduce the quarantining interval for retesting of the donor. This could facilitate logistics and reduce losses as quarantine FFP can be released earlier after donation and at regular donation intervals. Essential parameters for estimating the remaining infectious risks are the minimal infectious dose and replication kinetics of the viruses involved, the detection limits of the NAT tests applied, and the volume of plasma transfused. In essence it can be assumed that discontinuation of quarantining would only marginally increase the residual risk and that the reduction of the quarantine period to only 4 weeks would add an additional benefit to the viral safety of quarantine FFP.

Hepatites virais: um fator de risco na prática odontológica

During dental pratice, there is a potential hazard to patients and dentists of infectious diseases transmission. Those diseases include viral hepatitis due to its form of transmission and possible complications like liver cirrhosis and cancer. The hepatitis viruses of greatest relevance to dentistry are mainly hepatitis viruses B, C and D. There is increasing evidence that other hepatitis viruses as G, TT and SEN-V can be parenterally transmitted and also pose occupational hazards in dental pratice. Despite all the available knowledge, it is still possible to find professionals, professors and students which still neglect the biosafety procedures.

Do herpes e suas implicações audiológicas: uma revisao de literatura

TEMA: herpes e audiologia. OBJETIVO: realizar revisão teórica principalmente sobre os vírus herpes simples tipo 1, herpes simples tipo 2 e varicela-zoster, bem como sobre seus efeitos na audição humana. Esses se constituem nos tipos de vírus herpéticos humanos de maior relevância para a área da Audiologia dentro da ciência da Fonoaudiologia e, no entanto, são pouco conhecidos e estudados, especialmente no Brasil. MÉTODOS: realizou-se pesquisa em bases de dados eletrônicas nacionais e internacionais, incluindo SciELO, MEDLINE e LILACS, a partir da seguinte combinação de descritores: herpes simplex/zoster X hearing loss ou deafness. Foram selecionados estudos publicados desde a década de 90 até os dias atuais, relevando-se aqueles que contivessem maior valor informativo, contribuindo para os objetivos do presente trabalho. CONCLUSÃO: os vírus herpéticos estudados apresentam estreita relação com distúrbios auditivos, independentemente da idade em que o sujeito é acometido.

Allo-Reactive Potential of Multi-Virus Specific Cytotoxic T Cell Lines Recognizing Adenovirus Hexon, Cytomegalovirus pp65 and Epstein Barr Virus.

Abstract 5121 IntroductionCytomegalovirus (CMV), adenovirus (Ad), and Epstein-Barr virus (EBV) are major pathogens after allogeneic hematopoietic stem cell transplant. The current antiviral therapy is associated with significant myelosuppression, impaired hematopoietic reconstitution and consequently, higher rates of other infections. Adoptive immunotherapy utilizing allogeneic donor-derived virus specific cytotoxic T cell lines (CTL) has been shown to effectively treat refractory viral infections. Although such lines carry the theoretical risk of allo-reactivity and the resultant graft versus host disease, this complication has not been widely reported in recipients of single virus specific cell lines. To improve the efficiency of this therapeutic modality we have developed an approach to generate a single cell line that recognizes all three of these clinically relevant viruses while also having greatly reduced allo-reactive potential. MethodsPeripheral blood mononuclear cells (PBMC) from 3 healthy CMV seropositive donors were primed 3 times with autologous monocyte-derived dendritic cells pulsed with overlapping pools of pentadecapeptides spanning the antigen coding region of highly conserved regions of the Ad hexon protein and/or pools spanning the entire CMV pp65 protein. The lines were expanded for minimally one additional week using peptide loaded EBV transformed B cells (BLCL) as antigen presenting cells (APC). CMV, Ad and EBV anti-viral activity was monitored weekly by chromium release assay and the specificity of the lines was controlled by varying the peptide pools used for subsequent priming. The IFN-gamma ELISPOT assay was used to identify single peptides and to determine HLA restriction. The frequency and subset of single and pool peptide-reactive cells was determined using IFN-gamma intracellular cytokine production by flow cytometry. Fresh PBMC and established lines were additionally primed with fully allogeneic stimulators and response measured using intracellular cytokine production. ResultsEach of the 3 lines had robust proliferation and CTL activity against CMV and Ad peptide-pulsed targets. The lines consisted of 44.5-82.4% CD3+CD8+cells and 18.1-56.7% CD3+CD4+cells. CMV induced IFN-gamma response in 4.3-76.6% of CD3+CD8+cells and 0.4-47.5% of CD3+CD4+cells. Ad induced 1.4-43.9% of CD3+CD8+cells and 6.9-45.7% of CD3+CD4+cells to produce IFN-gamma. The 3 lines recognized 2-4 single CMV peptides and 2-6 single Ad peptides. The reactivity against these viral peptides was HLA restricted. At the time of testing none of the lines showed a strong EBV response. However, EBV reactivity could be detected with 1-2 additional primings, indicating that these precursors were present. Compared to fresh PBMC from the same donors, there was a 34.0±5.6 fold decrease in the frequency of alloreactive CD3+CD8+cells demonstrated in the intracellular cytokine assay. ConclusionsGeneration of multi-viral specific CTL in a single culture is feasible so long as antigen stimulation is controlled and this approach will greatly reduce the time, cost and number of cells required to restore viral immunity for patients receiving allogeneic stem cell transplant. The risk of allo-reactivity in these lines is greatly reduced compared to fresh PBMC. This makes adoptive cell therapy more practical, easier to implement and safer. DisclosuresNo relevant conflicts of interest to declare.

Caspase-Dependent Inhibition of Mousepox Replication by gzmB

BackgroundEctromelia virus is a natural mouse pathogen, causing mousepox. The cytotoxic T (Tc) cell granule serine-protease, granzyme B, is important for its control, but the underlying mechanism is unknown. Using ex vivo virus immune Tc cells, we have previously shown that granzyme B is able to activate several independent pro-apoptotic pathways, including those mediated by Bid/Bak/Bax and caspases-3/-7, in target cells pulsed with Tc cell determinants.Methods and FindingsHere we analysed the physiological relevance of those pro-apoptotic pathways in ectromelia infection, by incubating ectromelia-immune ex vivo Tc cells from granzyme A deficient (GzmB+ Tc cells) or granzyme A and granzyme B deficient (GzmA×B−/− Tc cell) mice with ectromelia-infected target cells. We found that gzmB-induced apoptosis was totally blocked in ectromelia infected or peptide pulsed cells lacking caspases-3/-7. However ectromelia inhibited only partially apoptosis in cells deficient for Bid/Bak/Bax and not at all when both pathways were operative suggesting that the virus is able to interfere with apoptosis induced by gzmB in case not all pathways are activated. Importantly, inhibition of viral replication in vitro, as seen with wild type cells, was not affected by the lack of Bid/Bak/Bax but was significantly reduced in caspase-3/-7-deficient cells. Both caspase dependent processes were strictly dependent on gzmB, since Tc cells, lacking both gzms, neither induced apoptosis nor reduced viral titers.SignificanceOut findings present the first evidence on the biological importance of the independent gzmB-inducible pro-apoptotic pathways in a physiological relevant virus infection model.

Viruses in subarctic lakes and their impact on benthic and pelagic bacteria

Virus-bacterium interactions were investigated in the pelagic and benthic habitats in a set of lakes along an altitudinal gradient in the subarctic northern Sweden. Viral and bacterial abundances showed a significant variation between the lakes, with the highest benthic microbial abundances recorded in a high-altitude lake [993 m above sea level (a.s.l.)], whereas the highest pelagic microbial abundances were found in a low-altitude lake (270 m a.s.l.). In the pelagic habitat, there was also a distinct difference in microbial abundances between the summer-autumn and the winter sampling occasion. A positive relationship was noted between viruses and bacteria in both the pelagic and the benthic habitats. Visibly virus-infected bacterial cells were uncommon in the pelagic habitat and undetectable in the benthos. Both lytic and lysogenic pelagic viral production rates were undetectable or low; thus, a possible explanation for the relative high viral abundances found in the water column could be an allochthonous input of viruses or release of sediment-derived viruses. Overall, our results provide novel information about the relevance of viruses in the subarctic region and indicate that viruses play only a minor role in the nutrient and carbon cycling in the microbial communities of subarctic lakes.

On the astrobiological relevance of viruses in extraterrestrial ecosystems

AbstractViruses are the dominant form of genetically reproducing entities on Earth. Yet, viruses are mostly neglected in the context of astrobiology due to their non-living nature. In this discussion it is considered whether viruses are likely to be transferred within bacterial endospores to other planetary bodies through lithopanspermia. Interestingly, it seems possible that ecosystems of panspermial origin might yield biospheres in which viruses are absent. The evolutionary pathway of life in these systems might differ significantly from the path observed on Earth. We hypothesize that the difference in the two potential ways for the emergence of life, those being panspermial or local origin, on any certain planet could be clarified by analyzing the diversity of virosphere on the planet. The use of viruses as putative biomarkers is also discussed and studied.

Structural Basis of Inhibition Specificities of 3C and 3C-like Proteases by Zinc-coordinating and Peptidomimetic Compounds

Human coxsackievirus (CV) belongs to the picornavirus family, which consists of over 200 medically relevant viruses. In picornavirus, a chymotrypsin-like protease (3Cpro) is required for viral replication by processing the polyproteins, and thus it is regarded as an antiviral drug target. A 3C-like protease (3CLpro) also exists in human coronaviruses (CoV) such as 229E and the one causing severe acute respiratory syndrome (SARS). To combat SARS, we previously had developed peptidomimetic and zinc-coordinating inhibitors of 3CLpro. As shown in the present study, some of these compounds were also found to be active against 3Cpro of CV strain B3 (CVB3). Several crystal structures of 3Cpro from CVB3 and 3CLpro from CoV-229E and SARS-CoV in complex with the inhibitors were solved. The zinc-coordinating inhibitor is tetrahedrally coordinated to the His40-Cys147 catalytic dyad of CVB3 3Cpro. The presence of specific binding pockets for the residues of peptidomimetic inhibitors explains the binding specificity. Our results provide a structural basis for inhibitor optimization and development of potential drugs for antiviral therapies.

Development and evaluation of a GFLV inverted repeat construct for genetic transformation of grapevine

Grapevine Fanleaf virus (GFLV) is one of the most damaging and widespread nepovirus in grapevine, causing the fanleaf disease. Here, we report the development of inverted repeat (IR) constructs, consisting of GFLV-derived sequences, for genetic transformation of grapevine to induce GFLV-specific silencing. The IR construct was designed with fragments of a conserved region of the GFLV movement protein (MPc) gene from a Tunisian isolate (GFLV tun) and with cassettes containing the neomycin phosphotransferase or bar gene as selectable marker. For proof of concept, the IR construct was transferred into Nicotiana benthamiana by Agrobacterium mediated transformation. Challenge inoculation of T1 transgenic plant lines with the relevant virus resulted in plants showing resistance, recovery, retarded infection and susceptibility. Northern blot analysis, using a virus-derived sequence (MPc) as probe, could detect transgene specific small interfering RNA in resistant transgenic N. benthamiana plants after GFLV inoculation. Embryogenic cells of grapevine (Vitis vinifera cv Arich dresse) were transformed with the IR construct in combination with the bar gene using A. tumefaciens strain LBA4404. After molecular characterization of regenerated putative transgenic grapevine plants, Northern blot analysis detected different levels of IR transcripts in independent transgenic grapevine lines indicating a post-transcriptional gene silencing.