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Christin Schmidt, Barbara S Schnierle Paul-Ehrlich-Institut, Department of Virology, Section AIDS and Newly Emerging Pathogens, Langen, GermanyCorrespondence: Barbara S Schnierle, Paul-Ehrlich-Institut, Department of Virology, Section AIDS and newly emerging pathogens, Paul-Ehrlich-Strasse 51.59, Langen, 63225, Germany, Tel/Fax +49 6103 77 5504, Email barbara.schnierle@pei.deAbstract: Chikungunya virus (CHIKV) is an alphavirus that has spread globally in the last twenty years. Although mortality is rather low, infection can result in debilitating arthralgia that can persist for years. Unfortunately, no treatments or preventive vaccines are currently licensed against CHIKV infections. However, a large range of promising preclinical and clinical vaccine candidates have been developed during recent years. This review will give an introduction into the biology of CHIKV and the immune responses that are induced by infection, and will summarize CHIKV vaccine development.Keywords: alphavirus, vector vaccines, inactivated virus, nucleic acid vaccines, immune response, arthralgia

A recombinant Ross River virus (RRV) that contains the fluorescent protein mCherry fused to the non-structural protein 3 (nsP3) was constructed, which allowed real-time imaging of viral replication. RRV-mCherry contained either the natural opal stop codon after the nsP3 gene or was constructed without a stop codon. The mCherry fusion protein did not interfere with the viral life cycle and deletion of the stop codon did not change the replication capacity of RRV-mCherry. Comparison of RRV-mCherry and chikungunya virus-mCherry infections, however, showed a cell type-dependent delay in RRV-mCherry replication in HEK 293T cells. This delay was not caused by differences in cell entry, but rather by an impeded nsP expression caused by the RRV inhibitor ZAP (zinc finger CCCH-Type, antiviral 1). The data indicate that viral replication of alphaviruses is cell-type dependent, and might be unique for each alphavirus.

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells promoting ACE2-mediated entry with and without TMPRSS2 coexpression. Electron microscopy and inhibitor experiments indicated TMPRSS2-mediated cell entry was associated with increased virion internalization into endosomes, and partially dependent upon clathrin-mediated endocytosis. TMPRSS2 increased panvariant uptake efficiency and enhanced early rates of virus replication, transcription, and secretion, with variant-specific profiles observed. On the host side, transcriptional profiling confirmed the magnitude of infection-induced antiviral and proinflammatory responses were linked to uptake efficiency, with TMPRSS2-assisted entry boosting early antiviral responses. In addition, TMPRSS2-enhanced infections increased rates of cytopathology, apoptosis, and necrosis and modulated virus secretion kinetics in a variant-specific manner. On the virus side, convergent signatures of cell-uptake-dependent innate immune induction were recorded in viral genomes, manifesting as switches in dominant coupled Nsp3 residues whose frequencies were correlated to the magnitude of the cellular response to infection. Experimentally, we demonstrated that selected Nsp3 mutations conferred enhanced interferon antagonism. More broadly, we show that TMPRSS2 orthologues from evolutionarily diverse mammals facilitate panvariant enhancement of cell uptake. In summary, our study uncovers previously unreported associations, linking cell entry efficiency to innate immune activation kinetics, cell death rates, virus secretion dynamics, and convergent selection of viral mutations. These data expand our understanding of TMPRSS2's role in the SARS-CoV-2 life cycle and confirm its broader significance in zoonotic reservoirs and animal models., Competing Interests: Competing interests statement:The authors declare no competing interest.

The present use of mRNA vaccines against COVID-19 has shown for the first time the potential of mRNA vaccines for infectious diseases. Here we will summarize the current knowledge about improved mRNA vaccines, i.e., the self-amplifying mRNA (saRNA) vaccines. This approach may enhance antigen expression by amplification of the antigen-encoding RNA. RNA design, RNA delivery, and the innate immune responses induced by RNA will be reviewed.

In the late 1970s, global vaccination programs resulted in the eradication of smallpox. The Monkeypox virus (MPXV), which is closely related to the smallpox-inducing variola virus, was previously endemic only in Sub-Saharan Africa but is currently spreading worldwide. Only older people who have been vaccinated against smallpox are expected to be sufficiently protected against poxviruses. Here I will summarize current knowledge about the virus, the disease caused by MPXV infections, and strategies to limit its spread.

Alphaviruses such as the human pathogenic chikungunya virus (CHIKV) and Ross River virus (RRV) can cause explosive outbreaks raising public health concerns. However, no vaccine or specific antiviral treatment is yet available. We recently established a CHIKV vaccine candidate based on trans-amplifying RNA (taRNA). This novel system consists of a replicase-encoding mRNA and a trans-replicon (TR) RNA encoding the antigen. The TR-RNA is amplified by the replicase in situ. We were interested in determining whether multiple TR-RNAs can be amplified in parallel and if, thus, a multivalent vaccine candidate can be generated. In vitro, we observed an efficient amplification of two TR-RNAs, encoding for the CHIKV and the RRV envelope proteins, by the replicase, which resulted in a high antigen expression. Vaccination of BALB/c mice with the two TR-RNAs induced CHIKV- and RRV-specific humoral and cellular immune responses. However, antibody titers and neutralization capacity were higher after immunization with a single TR-RNA. In contrast, alphavirus-specific T cell responses were equally potent after the bivalent vaccination. These data show the proof-of-principle that the taRNA system can be used to generate multivalent vaccines; however, further optimizations will be needed for clinical application.

The SARS-CoV-2 variant Omicron has spread world-wide and is responsible for rapid increases in infections, including in populations with high vaccination rates. Here, we analysed in the sera of vaccinated individuals the antibody binding to the receptor-binding domain (RBD) of the spike protein and the neutralization of wild-type (WT), Delta (B.1.617.2), and Omicron (B.1.1.529; BA.1) pseudotyped vectors. Although sera from individuals immunized with vector vaccines (Vaxzevria; AZ and COVID-19 Janssen, Ad26.COV2.S; J&J) were able to bind and neutralize WT and Delta, they showed only background levels towards Omicron. In contrast, mRNA (Comirnaty; BNT) or heterologous (AZ/BNT) vaccines induced weak, but detectable responses against Omicron. While RBD-binding antibody levels decreased significantly six months after full vaccination, the SARS-CoV-2 RBD-directed avidity remained constant. However, this still coincided with a significant decrease in neutralization activity against all variants. A third booster vaccination with BNT significantly increased the humoral immune responses against all tested variants, including Omicron. In conclusion, only vaccination schedules that included at least one dose of mRNA vaccine and especially an mRNA booster vaccination induced sufficient antibody levels with neutralization capacity against multiple variants, including Omicron.

The arthritogenic alphavirus, chikungunya virus (CHIKV), is now present in almost 100 countries worldwide. Further spread is very likely, which raises public health concerns. CHIKV infections cause fever and arthralgia, which can be debilitating and last for years. Here, we describe a CHIKV vaccine candidate based on trans -amplifying RNA (taRNA). The vaccine candidate consists of two RNAs: a non-replicating mRNA encoding for the CHIKV nonstructural proteins, forming the replicase complex and a trans -replicon (TR) RNA encoding the CHIKV envelope proteins. The TR-RNA can be amplified by the replicase in trans , and small RNA amounts can induce a potent immune response. The TR-RNA was efficiently amplified by the CHIKV replicase in vitro , leading to high protein expression, comparable to that generated by a CHIKV infection. In addition, the taRNA system did not recombine to replication-competent CHIKV. Using a prime-boost schedule, the vaccine candidate induced potent CHIKV-specific humoral and cellular immune responses in vivo in a mouse model. Notably, mice were protected against a high-dose CHIKV challenge infection with two vaccine doses of only 1.5 μg RNA. Therefore, taRNAs are a promising safe and efficient vaccination strategy against CHIKV infections., Competing Interests: U.S., T.B., and M.P. are inventors on patents and patent applications that cover parts of this article. U.S. is an employee at BioNTech Corporation (Mainz, Germany), a privately owned company developing therapeutic RNA. All others declare no competing interests., (© 2022 The Authors.)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has now been continuing for more than two years. The infection causes COVID-19, a disease of the respiratory and cardiovascular system of variable severity. Here, the humoral immune response of 80 COVID-19 patients from the University Hospital Frankfurt/Main, Germany, was characterized longitudinally. The SARS-CoV-2 neutralization activity of serum waned over time. The neutralizing potential of serum directed towards the human alpha-coronavirus NL-63 (NL63) also waned, indicating that no cross-priming against alpha-coronaviruses occurred. A subset of the recovered patients ( n = 13) was additionally vaccinated with the mRNA vaccine Comirnaty. Vaccination increased neutralization activity against SARS-CoV-2 wild-type (WT), Delta, and Omicron, although Omicron-specific neutralization was not detectable prior to vaccination. In addition, the vaccination induced neutralizing antibodies against the more distantly related SARS-CoV-1 but not against NL63. The results indicate that although SARS-CoV-2 humoral immune responses induced by infection wane, vaccination induces a broad neutralizing activity against multiple SARS-CoVs, but not to the common cold alpha-coronavirus NL63.

In light of an increasing number of vaccinated and convalescent individuals, there is a major need for the development of robust methods for the quantification of neutralizing antibodies; although, a defined correlate of protection is still missing. Sera from hospitalized COVID-19 patients suffering or not suffering from acute respiratory distress syndrome (ARDS) were comparatively analyzed by plaque reduction neutralization test (PRNT) and pseudotype-based neutralization assays to quantify their neutralizing capacity. The two neutralization assays showed comparable data. In case of the non-ARDS sera, there was a distinct correlation between the data from the neutralization assays on the one hand, and enzyme-linked immune sorbent assay (ELISA), as well as biophysical analyses, on the other hand. As such, surface plasmon resonance (SPR)-based assays for quantification of binding antibodies or analysis of the stability of the antigen-antibody interaction and inhibition of syncytium formation, determined by cell fusion assays, were performed. In the case of ARDS sera, which are characterized by a significantly higher fraction of RBD-binding IgA antibodies, there is a clear correlation between the neutralization assays and the ELISA data. In contrast to this, a less clear correlation between the biophysical analyses on the one hand and ELISAs and neutralization assays on the other hand was observed, which might be explained by the heterogeneity of the antibodies. To conclude, for less complex immune sera-as in cases of non-ARDS sera-combinations of titer quantification by ELISA with inhibition of syncytium formation, SPR-based analysis of antibody binding, determination of the stability of the antigen-antibody complex, and competition of the RBD-ACE2 binding represent alternatives to the classic PRNT for analysis of the neutralizing potential of SARS-CoV-2-specific sera, without the requirement for a BSL3 facility.

Alphaviruses have a single-stranded, positive-sense RNA genome that contains two open reading frames encoding either the non-structural or the structural genes. Upon infection, the genomic RNA is translated into the non-structural proteins (nsPs). NsPs are required for viral RNA replication and transcription driven from the subgenomic promoter (sgP). Transfection of an RNA encoding the luciferase gene under the control of the sgP into cells enabled the detection of replication-competent chikungunya virus (CHIKV) or Mayaro virus (MAYV) with high sensitivity as a function of the induced luciferase activity. This assay principle was additionally used to analyze virus-neutralizing antibodies in sera and might be an alternative to standard virus neutralization assays based on virus titration or the use of genetically modified tagged viruses.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and more than a million deaths. The infection causes COVID-19, a disease of the respiratory system of divergent severity. No treatment exists. Epigallocatechin-3-gallate (EGCG), the major component of green tea, has several beneficial properties, including antiviral activities. Therefore, we examined whether EGCG has antiviral activity against SARS-CoV-2. EGCG blocked not only the entry of SARS-CoV-2, but also MERS- and SARS-CoV pseudotyped lentiviral vectors and inhibited virus infections in vitro . Mechanistically, inhibition of the SARS-CoV-2 spike-receptor interaction was observed. Thus, EGCG might be suitable for use as a lead structure to develop more effective anti-COVID-19 drugs.

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has caused a pandemic with tens of millions of cases and hundreds of thousands of deaths. The infection causes coronavirus disease 2019 (COVID-19), a disease of the respiratory system of divergent severity. In the current study, humoral immune responses were characterized in a cohort of 143 patients with COVID-19 from the University Hospital Frankfurt am Main, Germany., Methods: SARS-CoV-2-specific-antibodies were detected by enzyme-linked immunosorbent assay (ELISA). SARS-CoV-2 and human coronavirus NL63 neutralization activity was analyzed with pseudotyped lentiviral vectors., Results: The severity of COVID-19 increased with age, and male patients encountered more serious symptoms than female patients. Disease severity was correlated with the amount of SARS-CoV-2-specific immunoglobulin (Ig) G and IgA and the neutralization activity of the antibodies. The amount of SARS-CoV-2-specific IgG antibodies decreased with time after polymerase chain reaction conformation of the infection, and antibodies directed against the nucleoprotein waned faster than spike protein-directed antibodies. In contrast, for the common flu coronavirus NL63, COVID-19 disease severity seemed to be correlated with low NL63-neutralizing activities, suggesting the possibility of cross-reactive protection., Conclusion: The results describe the humoral immune responses against SARS-CoV-2 and might aid the identification of correlates of protection needed for vaccine development., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and has spread worldwide, with millions of cases and more than 1 million deaths to date. The gravity of the situation mandates accelerated efforts to identify safe and effective vaccines. Here, we generated measles virus (MeV)-based vaccine candidates expressing the SARS-CoV-2 spike glycoprotein (S). Insertion of the full-length S protein gene in two different MeV genomic positions resulted in modulated S protein expression. The variant with lower S protein expression levels was genetically stable and induced high levels of effective Th1-biased antibody and T cell responses in mice after two immunizations. In addition to neutralizing IgG antibody responses in a protective range, multifunctional CD8 + and CD4 + T cell responses with S protein-specific killing activity were detected. Upon challenge using a mouse-adapted SARS-CoV-2, virus loads in vaccinated mice were significantly lower, while vaccinated Syrian hamsters revealed protection in a harsh challenge setup using an early-passage human patient isolate. These results are highly encouraging and support further development of MeV-based COVID-19 vaccines., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Background: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes severe flu-like symptoms. The acute symptoms disappear after 1 week, but chronic arthralgia can persist for years. In this study, humoral immune responses in CHIKV-infected patients and vaccinees were analyzed., Methods: Alphavirus neutralization activity was analyzed with pseudotyped lentiviral vectors, and antibody epitope mapping was performed with a peptide array., Results: The greatest CHIKV neutralization activity was observed 60-92 days after onset of symptoms. The amount of CHIKV-specific antibodies and their binding avidity and cross-reactivity with other alphaviruses increased over time. Chikungunya virus and o'nyong-nyong virus (ONNV) were both neutralized to a similar extent. Linear antibody binding epitopes were mainly found in E2 domain B and the acid-sensitive regions (ASRs). In addition, serum samples from healthy volunteers vaccinated with a measles-vectored chikungunya vaccine candidate, MV-CHIK, were analyzed. Neutralization activity in the samples from the vaccine cohort was 2- to 6-fold lower than in samples from CHIKV-infected patients. In contrast to infection, vaccination only induced cross-neutralization with ONNV, and the E2 ASR1 was the major antibody target., Conclusions: These data could assist vaccine design and enable the identification of correlates of protection necessary for vaccine efficacy., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

FHL1 has been identified as a host protein that is essential for chikungunya virus (CHIKV) replication. FHL1 interacts with the chikungunya non-structural protein 3, which is thought to recruit cellular proteins to the viral replication complex. Inhibition of this interaction is a promising target for drug development., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Chikungunya virus infection causes a debilitating febrile illness that in many affected individuals is associated with long-term sequelae that can persist for months or years. Over the past decade a large number of candidate vaccines have been developed, several of which have now entered clinical trials. The rapid and sporadic nature of chikungunya outbreaks poses challenges for planning of large clinical efficacy trials suggesting that licensure of chikungunya vaccines may utilize non-traditional approval pathways based on identification of immunological endpoint(s) predictive of clinical benefit. This report reviews the current status of nonclinical and clinical testing and potential challenges for defining a suitable surrogate or correlate of protection., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Chikungunya virus (CHIKV) is clinically the most relevant member of the Alphavirus genus. Like alphaviruses in general, CHIKV has the capacity to infect a large variety of cells, tissues, and species. This broad host tropism of CHIKV indicates that the virus uses a ubiquitously expressed receptor to infect cells. This review summarizes the current knowledge available on cellular CHIKV receptors and the attachment factors used by CHIKV.

Attenuated poxviruses like modified vaccinia virus Ankara (MVA) are promising vectors for vaccines against infectious diseases and cancer. However, host innate immune responses interfere with the viral life cycle and also influence the immunogenicity of vaccine vectors. Sterile alpha motif (SAM) domain and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a phosphohydrolase and reduces cellular deoxynucleoside triphosphate (dNTP) concentrations, which impairs poxviral DNA replication in human dendritic cells (DCs). Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) encode an accessory protein called viral protein X (Vpx) that promotes proteasomal degradation of SAMHD1, leading to a rapid increase in cellular dNTP concentrations. To study the function of SAMHD1 during MVA infection of human DCs, the SIV vpx gene was introduced into the MVA genome (resulting in recombinant MVA-vpx). Infection of human DCs with MVA-vpx led to SAMHD1 protein degradation and enabled MVA-vpx to replicate its DNA genome and to express genes controlled by late promoters. Late gene expression by MVA-vpx might improve its vaccine vector properties; however, type I interferon expression was unexpectedly blocked by Vpx-expressing MVA. MVA-vpx can be used as a tool to study poxvirus-host interactions and vector safety. IMPORTANCE SAMHD1 is a phosphohydrolase and reduces cellular dNTP concentrations, which impairs poxviral DNA replication. The simian SIV accessory protein Vpx promotes degradation of SAMHD1, leading to increased cellular dNTP concentrations. Vpx addition enables poxviral DNA replication in human dendritic cells (DCs), as well as the expression of viral late proteins, which is normally blocked. SAMHD1 function during modified vaccinia virus Ankara (MVA) infection of human DCs was studied with recombinant MVA-vpx expressing Vpx. Infection of human DCs with MVA-vpx decreased SAMHD1 protein amounts, enabling MVA DNA replication and expression of late viral genes. Unexpectedly, type I interferon expression was blocked after MVA-vpx infection. MVA-vpx might be a good tool to study SAMHD1 depletion during poxviral infections and to provide insights into poxvirus-host interactions., (Copyright © 2019 American Society for Microbiology.)

Background: Alphaviruses are transmitted by arthropod vectors and can be found worldwide. Alphaviruses of the Semliki Forest complex such as chikungunya virus (CHIKV), Mayaro virus (MAYV) or Ross River virus (RRV) cause acute febrile illness and long-lasting arthralgia in humans, which cannot be clinically discriminated from a dengue virus or Zika virus infection. Alphaviruses utilize a diverse array of mosquito vectors for transmission and spread. For instance, adaptation of CHIKV to transmission by Aedes albopictus has increased its spread and resulted in large outbreaks in the Indian Ocean islands. For many alphaviruses commercial diagnostic tests are not available or show cross-reactivity among alphaviruses. Climate change and globalization will increase the spread of alphaviruses and monitoring of infections is necessary and requires virus-specific methods., Method: We established an alphavirus neutralization assay in a 384-well format by using pseudotyped lentiviral vectors., Results: MAYV-specific reactivity could be discriminated from CHIKV reactivity. Human plasma from blood donors infected with RRV could be clearly identified and did not cross-react with other alphaviruses., Conclusion: This safe and easy to use multiplex assay allows the discrimination of alphavirus-specific reactivity within a single assay and has potential for epidemiological surveillance. It might also be useful for the development of a pan-alphavirus vaccine., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Silvestrol, a natural compound that is isolated from plants of the genus Aglaia , is a specific inhibitor of the RNA helicase eIF4A, which unwinds RNA secondary structures in 5'-untranslated regions (UTRs) of mRNAs and allows translation. Silvestrol has a broad antiviral activity against multiple RNA virus families. Here, we show that silvestrol inhibits the replication of chikungunya virus (CHIKV), a positive single-stranded RNA virus. Silvestrol delayed the protein synthesis of non-structural (nsPs) and structural proteins, resulting in a delayed innate response to CHIKV infection. Interferon-α induced STAT1 phosphorylation was not inhibited nor did eIF2α become phosphorylated 16 h post infection in the presence of silvestrol. In addition, the host protein shut-off induced by CHIKV infection was decreased in silvestrol-treated cells. Silvestrol acts by limiting the amount of nsPs, and thereby reducing CHIKV RNA replication. From our results, we propose that inhibition of the host helicase eIF4A might have potential as a therapeutic strategy to treat CHIKV infections.

Background: With the ongoing public health emergency due to Zika virus (ZIKV), nucleic acid testing (NAT) is essential for clinical diagnosis and screening of blood donors. However, NAT for ZIKV has not been standardized, and this study was performed to establish a World Health Organization (WHO) International Standard (IS) for ZIKV RNA; WHO ISs have been used to improve detection and quantification of blood-borne viruses., Study Design and Methods: The candidate IS (cIS), code number 11468/16, was prepared by heat inactivation and lyophilization of a ZIKV strain isolated from a patient in French Polynesia in 2013. The cIS was evaluated together with other reference materials, including both Asian and African ZIKV lineages as well as a panel of clinical samples and in vitro-transcribed RNAs. The samples for evaluation were distributed to 24 laboratories from 11 countries. The assays used consisted of a mixture of in-house developed and commercial assays (available or in development)., Results: The potencies of the standards were determined by quantitative and qualitative assays. In total, 37 sets of data were analyzed: 19 from quantitative assays and 18 from qualitative assays. Data demonstrated wide variations in reported potencies of the cIS and the other study samples., Conclusions: Assay variability was substantially reduced when ZIKV RNA concentrations from the biological reference materials and clinical samples were expressed relative to the cIS. Thus, the WHO has established 11468/16 as the 1 st IS for ZIKV RNA, with a unitage of 50,000,000 IU/mL., (© 2017 AABB.)

Background: Zika virus (ZIKV) is an emerging mosquito-borne Flavivirus of major public health concern. The potential for ZIKV transmission by blood transfusion has been demonstrated; however, inactivation or removal of ZIKV during the manufacture of plasma-derived medicinal products has not been specifically investigated., Study Design and Methods: Inactivation of ZIKV by pasteurization and solvent/detergent (S/D) treatment was investigated by spiking high-titer ZIKV stocks into human serum albumin and applying either heat or adding different mixtures of S/D reagents and assaying for infectious virus particles. Removal of ZIKV was evaluated using filters of differing pore sizes (75, 40, 35, and 19 nm), assaying for infectious virus and RNA. Electron microscopy was performed to determine the size of ZIKV particles. Neutralization of virus infectivity by immunoglobulins was investigated., Results: ZIKV was effectively and rapidly inactivated by liquid heat treatment as well as by various mixtures of S/D reagents with reduction factors more than 4 log, in each case. Effective reduction of ZIKV infectivity was demonstrated for virus filtration for filters with average pore sizes of not more than 40 nm, although a significant proportion of virus RNA was detected in the 40- to 35-nm filtrates likely due to the presence of subviral particles observed by electron microscopy. None of the immunoglobulin preparations investigated neutralized ZIKV infectivity., Conclusions: Pasteurization and S/D treatment very rapidly inactivated ZIKV and filters with a pore size of not more than 40 nm removed all infectious ZIKV, demonstrating the effectiveness of these virus reduction strategies used during the manufacture of plasma-derived medicinal products., (© 2016 The Authors Transfusion published by Wiley Periodicals, Inc. on behalf of AABB.)

Background: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions, including Europe and the United States of America. In the past, CHIKV has mainly affected developing countries, but has recently caused large outbreaks in the Caribbean and Latin America. No treatment or licensed CHIKV vaccine exists., Methodology/principal Findings: Here, we have identified determinants in the CHIKV cell-attachment protein E2 that facilitate cell binding. The extracellular part of the E2 gene is subdivided into the three domains, A, B, and C. These domains were expressed in E. coli and as Fc-fusion proteins generated from HEK293T cells and used for cell-binding assays. Domains A and B bound to all cells tested, independently of their permissiveness to CHIKV infection. Domain C did not bind to cells at all. Furthermore, CHIKV cell entry was promoted by cell-surface glycosaminoglycans (GAGs) and domain B interacted exclusively with GAG-expressing cells. Domain A also bound, although only moderately, to GAG-deficient cells. Soluble GAGs were able to inhibit CHIKV infection up to 90%; however, they enhanced the transduction rate of CHIKV Env pseudotyped vectors in GAG-negative cells., Conclusion/significance: These data imply that CHIKV uses at least two mechanisms to enter cells, one GAG-dependent, via initial attachment through domain B, and the other GAG-independent, via attachment of domain A. These data give indications that CHIKV uses multiple mechanisms to enter cells and shows the potential of GAGs as lead structures for developing antiviral drugs., Competing Interests: The authors have declared that no competing interests exist.

Background: Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes high fever, rash, and recurrent arthritis in humans. It has efficiently adapted to Aedes albopictus, which also inhabits temperate regions and currently causes large outbreaks in the Caribbean and Latin America. Ebola virus (EBOV) is a member of the filovirus family. It causes the Ebola virus disease (EDV), formerly known as Ebola hemorrhagic fever in humans and has a mortality rate of up to 70 %. The last outbreak in Western Africa was the largest in history and has caused approximately 25,000 cases and 10,000 deaths. For both viral infections no specific treatment or licensed vaccine is currently available. The bis-hexasulfonated naphthylurea, suramin, is used as a treatment for trypanosome-caused African river blindness. As a competitive inhibitor of heparin, suramin has been described to have anti-viral activity., Methods: We tested the activity of suramin during CHIKV or Ebola virus infection, using CHIKV and Ebola envelope glycoprotein pseudotyped lentiviral vectors and wild-type CHIKV and Ebola virus., Results: Suramin efficiently inhibited CHIKV and Ebola envelope-mediated gene transfer while vesicular stomatitis virus G protein pseudotyped vectors were only marginally affected. In addition, suramin was able to inhibit wild-type CHIKV and Ebola virus replication in vitro. Inhibition occurred at early time points during CHIKV infection., Conclusion: Suramin, also known as Germanin or Bayer-205, is a market-authorized drug, however shows significant side effects, which probably prevents its use as a CHIKV drug, but due to the high lethality of Ebola virus infections, suramin might be valuable against Ebola infections.

Myxobacteria produce secondary metabolites many of which were described to have various biological effects including anti-fungal, anti-bacterial and anti-viral activity. The majority of these metabolites are novel scaffolds with unique modes-of-action and hence might be potential leads for drug discovery. Here, we tested a myxobacterial natural product library for compounds with inhibitory activity against Ebola virus (EBOV). The assay was performed with a surrogate system using Ebola envelope glycoprotein (GP) pseudotyped lentiviral vectors. EBOV specificity was proven by counter-screening with vesicular stomatitis virus G protein pseudotyped vectors. Two compounds were identified that preferentially inhibited EBOV GP mediated cell entry: Chondramides that act on the actin skeleton but might be too toxic and noricumazole A, a potassium channel inhibitor, which might constitute a novel pathway to inhibit Ebola virus cell entry., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever, and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions including Europe and the United States of America. CHIKV has recently caused large outbreaks in Latin America. No treatment or licensed CHIKV vaccine exists. Traditional medicines are known to have anti-viral effects; therefore, we examined whether curcumin or Boswellia serrata gum resin extract have antiviral activity against CHIKV. Both compounds blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV infection in vitro. In addition, vesicular stomatitis virus vector particles and viral infections were also inhibited to the same extent, indicating a broad antiviral activity. Although the bioavailability of these compounds is rather poor, they might be used as a lead structure to develop more effective antiviral drugs or might be used topically to prevent CHIKV spread in the skin after mosquito bites., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The mosquito-borne Chikungunya virus (CHIKV) causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses., Methodology/principal Findings: E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L) and surface-exposed parts of the E2 domain A (sA) alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+) was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+) induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA), MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice., Conclusions/significance: The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever and severe arthritis that can persist for years. CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions, including Europe and the United States of America and might cause new, large outbreaks there. No treatment or licensed CHIKV vaccine exists. Epigallocatechin-3-gallate (EGCG), the major component of green tea, has, among other beneficial properties, antiviral activities. Therefore, we examined if EGCG has antiviral activity against CHIKV. EGCG inhibited CHIKV infection in vitro, blocked entry of CHIKV Env-pseudotyped lentiviral vectors and inhibited CHIKV attachment to target cells. Thus EGCG might be used as a lead structure to develop more effective antiviral drugs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Chikungunya virus (CHIKV) is a mosquito-transmitted Alphavirus that causes chikungunya fever and has infected millions of people mainly in developing countries. The associated disease is characterized by rash, high fever and severe arthritis that can persist for years. Since the epidemic on La Réunion in 2006, CHIKV has adapted to Aedes albopictus, which also inhabits temperate regions of the eastern and western hemispheres, including Europe and the United States. A. albopictus might continue migrating north with continuing climate change and CHIKV would then no longer be confined to the developing nations. No treatment or licensed CHIKV vaccine exists. A CHIKV neutralization assay in a 384-well format by using CHIKV-pseudotyped lentiviral vectors was established. This assay system can be used for entry inhibitor screening under a reduced safety level (S2). Production of CHIKV-pseudotyped lentiviral vectors and the reaction volume are optimized. A dose dependent, specific neutralization of CHIKV-pseudotyped vectors with sera of CHIKV-infected individuals could be measured in a 384-well format. A safe and simple multiplex assay for the analysis of CHIKV neutralizing activities was developed and will be able to improve drug and vaccine development as well as it would improve the understanding of CHIKV epidemics regarding antibody responses., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Human endogenous retroviruses (HERVs) are remnants of ancestral infections and chromosomally integrated in all cells of an individual, are transmitted only vertically and are defective in viral replication. However enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed inter-alia in HIV-infected individuals and tumor patients. Therefore HERV-K might serve as a tumor-specific antigen or even as a constant target for the development of an HIV vaccine., Results: To verify our hypothesis, we tested the immunogenicity of HERV-K Gag by using a recombinant vaccinia virus (MVA-HKcon) expressing the HERV-K Gag protein and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) and the HERV-K Gag protein (RLZ-HKGag cells). Subcutaneous application of RLZ-HKGag cells into syngenic BALB/c mice resulted in the formation of local tumors in MVA vaccinated mice. MVA-HKcon vaccination reduced the tumor growth. Furthermore, intravenous injection of RLZ-HKGag cells led to the formation of pulmonary metastases. Vaccination of tumor-bearing mice with MVA-HKcon drastically reduced the number of pulmonary RLZ-HKGag tumor nodules compared to vaccination with wild-type MVA., Conclusion: The data demonstrate that HERV-K Gag is a useful target for vaccine development and might offer new treatment opportunities for cancer patients.

Human endogenous retrovirus (HERV) genomes are chromosomally integrated in all cells of an individual. They are normally transcriptionally silenced and transmitted only vertically. Enhanced expression of HERV-K accompanied by the emergence of anti-HERV-K-directed immune responses has been observed in tumor patients and HIV-infected individuals. As HERV-K is usually not expressed and immunological tolerance development is unlikely, it is an appropriate target for the development of immunotherapies. We generated a recombinant vaccinia virus (MVA-HKenv) expressing the HERV-K envelope glycoprotein (ENV), based on the modified vaccinia virus Ankara (MVA), and established an animal model to test its vaccination efficacy. Murine renal carcinoma cells (Renca) were genetically altered to express E. coli beta-galactosidase (RLZ cells) or the HERV-K ENV gene (RLZ-HKenv cells). Intravenous injection of RLZ-HKenv cells into syngenic BALB/c mice led to the formation of pulmonary metastases, which were detectable by X-gal staining. A single vaccination of tumor-bearing mice with MVA-HKenv drastically reduced the number of pulmonary RLZ-HKenv tumor nodules compared to vaccination with wild-type MVA. Prophylactic vaccination of mice with MVA-HKenv precluded the formation of RLZ-HKenv tumor nodules, whereas wild-type MVA-vaccinated animals succumbed to metastasis. Protection from tumor formation correlated with enhanced HERV-K ENV-specific killing activity of splenocytes. These data demonstrate for the first time that HERV-K ENV is a useful target for vaccine development and might offer new treatment opportunities for diverse types of cancer.

Haematopoietic humanization of mice is used frequently to study the human immune system and its reaction upon experimental intervention. Immunocompromised non-obese diabetic (NOD)-Rag1(-/-) mice, additionally deficient for the common gamma chain of cytokine receptors (γc) (NOD-Rag1(-/-) γc(-/-) mice), lack B, T and natural killer (NK) cells and allow for efficient human peripheral mononuclear cell (PBMC) engraftment. However, a major experimental drawback for studies using these mice is the rapid onset of graft-versus-host disease (GVHD). In order to elucidate the contribution of the xenogenic murine major histocompatibility complex (MHC) class II in this context, we generated immunodeficient mice expressing human MHC class II [human leucocyte antigen (HLA)-DQ8] on a mouse class II-deficient background (Aβ(-/-) ). We studied repopulation and onset of GVHD in these mouse strains following transplantation of DQ8 haplotype-matched human PBMCs. The presence of HLA class II promoted the repopulation rates significantly in these mice. Virtually all the engrafted cells were CD3(+) T cells. The presence of HLA class II did not advance B cell engraftment, such that humoral immune responses were undetectable. However, the overall survival of DQ8-expressing mice was prolonged significantly compared to mice expressing mouse MHC class II molecules, and correlated with an increased time span until onset of GVHD. Our data thus demonstrate that this new mouse strain is useful to study GVHD, and the prolonged animal survival and engraftment rates make it superior for experimental intervention following PBMC engraftment., (© 2013 British Society for Immunology.)

Background/aim: The cell line GH was established from germ-cell tumor tissue; human endogenous retrovirus-K (HERV-K) expression was detectable after prolonged culture of the cells, particularly in cells that formed domes and vesicles. In addition, keeping GH cells in culture at high cell densities increased HERV-K expression. Here, we studied whether this inducible HERV-K expression is accompanied by differences in microRNA (miRNA) expression patterns of GH cells., Materials and Methods: The global miRNA expression pattern of GH cell samples (HERV-K high versus low) was analyzed by miRNA arrays., Results: Two miRNAs were found to be differentially regulated and to exhibit expression parallel to that of HERV-K. The identified miRNAs-663 and -638, have been reported to be involved in multiple processes, including cellular senescence. However, induction of HERV-K expression did not change the cellular senescence status of GH cells., Conclusion: The expression of these two miRNAs might be useful as novel diagnostic and prognostic markers in patients with tumors.

Interferon-stimulated genes fulfill innate antiviral effector functions. Among them, tetherin (THN) blocks the release of many enveloped viruses from infected cells. Vaccinia virus (VACV) encodes immune modulators interfering with antiviral host responses. Therefore, it was tempting to study a potential VACV-THN interaction. Remarkably, THN expression did not inhibit VACV release and replication. VACV infection did not diminish THN surface levels or impair its function on retroviral release. This suggests that THN is unable to restrict VACV replication.

Background: Viral genomes of the human endogenous retrovirus K (HERV-K) family are integrated into the human chromosome and are transmitted vertically as Mendelian genes. Although viral particles are released by some transformed cells, they have never been shown to be infectious. In general, gammaretroviruses are produced as immature viral particles by accumulation of the Gag polyproteins at the plasma membrane, which subsequently bud from the cell surface. After release from the cell, Gag is further processed by proteolytic cleavage by the viral protease (PR), which results in morphologically mature particles with condensed cores. The HERV-K Gag polyprotein processing and function has not yet been precisely determined., Results: We generated a recombinant poxvirus, encoding the human endogenous retrovirus K consensus gag-pro-pol genes (MVA-HERV-Kcon) and obtained high levels of HERV-K Gag expression. The resulting retroviral particle assembled at the plasma membrane, as is typical for gammaretroviruses; and immature as well as mature retrovirus-like particles (VLPs) were observed around the infected cells. VLPs were purified, concentrated and separated by two-dimensional gel electrophoresis. The HERV-K Gag fragments were identified by mass spectroscopy and N-terminal sequencing which revealed that HERV-K Gag is processed into MA, a short spacer peptide, p15, CA and NC., Conclusion: The cleavage sites of HERV-K Gag were mapped and found to be highly conserved among HERV-K genomes. The consensus HERV-K gag gene used in this study is known to support viral, infectivity 1, and thus the cleavage sites that were mapped in this study for all the Gag components are relevant for HERV-K infectivity., (© 2011 Kraus et al; licensee BioMed Central Ltd.)

RNA interference (RNAi) technology has not only become a powerful tool for functional genomics, but also allows rapid drug target discovery and in vitro validation of these targets in cell culture. Furthermore, RNAi represents a promising novel therapeutic option for treating human diseases, in particular cancer. Selective gene silencing by RNAi can be achieved essentially by two nucleic acid based methods: i) cytoplasmic delivery of short double-stranded (ds) interfering RNA oligonucleotides (siRNA), where the gene silencing effect is only transient in nature, and possibly not suitable for all applications; or ii) nuclear delivery of gene expression cassettes that express short hairpin RNA (shRNA), which are processed like endogenous interfering RNA and lead to stable gene down-regulation. Both processes involve the use of nucleic acid based drugs, which are highly charged and do not cross cell membranes by free diffusion. Therefore, in vivo delivery of RNAi therapeutics must use technology that enables the RNAi therapeutic to traverse biological membrane barriers in vivo. Viruses and the vectors derived from them carry out precisely this task and have become a major delivery system for shRNA. Here, we summarize and compare different currently used viral delivery systems, give examples of in vivo applications, and indicate trends for new developments, such as replicating viruses for shRNA delivery to cancer cells.

Cutaneous T-cell lymphomas (CTCL) are characterised by clonal expansion of helper T lymphocytes that infiltrate the skin. Only a small number of cell lines exist to study cellular pathways leading to T-cell transformation and to identify new targets for intervention. We wanted to investigate the inhibition of mTOR as a possible therapeutic target in CTCL. Primary cells of patients with Sézary syndrome (SS) and conventional CTCL cell lines were analysed. Constitutive activation of mTOR was found, and concomitantly, we could show that rapamycin, a specific inhibitor of mTOR, inhibits CTCL cell growth in vitro by induction of cell cycle arrest. Using a previously established animal model for CTCL, we additionally observed upon rapamycin treatment tumor growth inhibition in vivo. In summary, primary cells from patients with SS as well as CTCL cell lines allowed us to identify mTOR as an important target for intervention.

The human immunodeficiency virus type 1 (HIV-1) coreceptor use and viral evolution were analyzed in blood samples from an HIV-1 infected patient undergoing allogeneic stem cell transplantation (SCT). Coreceptor use was predicted in silico from sequence data obtained from the third variable loop region of the viral envelope gene with two software tools. Viral diversity and evolution was evaluated on the same samples by Bayesian inference and maximum likelihood methods. In addition, phenotypic analysis was done by comparison of viral growth in peripheral blood mononuclear cells and in a CCR5 (R5)-deficient T-cell line which was controlled by a reporter assay confirming viral tropism. In silico coreceptor predictions did not match experimental determinations that showed a consistent R5 tropism. Anti-HIV directed antibodies could be detected before and after the SCT. These preexisting antibodies did not prevent viral rebound after the interruption of antiretroviral therapy during the SCT. Eventually, transplantation and readministration of anti-retroviral drugs lead to sustained increase in CD4 counts and decreased viral load to undetectable levels. Unexpectedly, viral diversity decreased after successful SCT. Our data evidence that only R5-tropic virus was found in the patient before and after transplantation. Therefore, blocking CCR5 receptor during stem cell transplantation might have had beneficial effects and this might apply to more patients undergoing allogeneic stem cell transplantation. Furthermore, we revealed a scenario of HIV-1 dynamic different from the commonly described ones. Analysis of viral evolution shows the decrease of viral diversity even during episodes with bursts in viral load.

Highly pathogenic avian influenza viruses of the H5N1 subtype are responsible for an increasing number of infections in humans since 2003. More than 60% of the infections is lethal and new infections are reported frequently. In the light of the pandemic threat caused by these events the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the HA gene of an influenza A/H5N1 virus is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous influenza A/H5N1 viruses in mice. We also evaluated the recombinant MVA vector expressing the HA of influenza A/H5N1 virus A/Vietnam/1194/04 (MVA-HA-VN/04) in non-human primates. Cynomolgus macaques were immunized twice and then challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity. Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia. Therefore MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic avian influenza A/H5N1 viruses.

Modified vaccinia virus Ankara (MVA) is a highly attenuated and replication-deficient vaccinia virus (VACV) that is being evaluated as replacement smallpox vaccine and candidate viral vector. MVA lacks many genes associated with virulence and/or regulation of virus tropism. The 68-kDa ankyrin-like protein (68k-ank) is the only ankyrin repeat-containing protein that is encoded by the MVA genome and is highly conserved throughout the Orthopoxvirus genus. We showed previously that 68k-ank is composed of ankyrin repeats and an F-box-like domain and forms an SCF ubiquitin ligase complex together with the cellular proteins Skp1a and Cullin-1. We now report that 68k-ank (MVA open reading frame 186R) is an essential factor for completion of the MVA intracellular life cycle in nonpermissive human and murine cells. Infection of mouse NIH 3T3 and human HaCaT cells with MVA with a deletion of the 68k-ank gene (MVA-Delta68k-ank) was characterized by an extensive reduction of viral intermediate RNA and protein, as well as late transcripts and drastically impaired late protein synthesis. Furthermore, infections with MVA-Delta68k-ank failed to induce the host protein shutoff that is characteristic of VACV infections. Although we demonstrated that proteasome function in general is essential for the completion of the MVA molecular life cycle, we found that a mutant 68k-ank protein with a deletion of the F-box-like domain was able to fully complement the deficiency of MVA-Delta68k-ank to express all classes of viral genes. Thus, our data demonstrate that the 68k-ank protein contains another critical domain that may function independently of SCF ubiquitin ligase complex formation, suggesting multiple activities of this interesting regulatory protein.

Background: Highly pathogenic avian influenza viruses of the H5N1 subtype have been responsible for an increasing number of infections in humans since 2003. More than 60% of infected individuals die, and new infections are reported frequently. In light of the pandemic threat caused by these events, the rapid availability of safe and effective vaccines is desirable. Modified vaccinia virus Ankara (MVA) expressing the hemagglutinin (HA) gene of H5N1 viruses is a promising candidate vaccine that induced protective immunity against infection with homologous and heterologous H5N1 influenza virus in mice., Methods: In the present study, we evaluated a recombinant MVA vector expressing the HA gene of H5N1 influenza virus A/Vietnam/1194/04 (MVA-HA-VN/04) in nonhuman primates. Cynomolgus macaques were immunized twice and then were challenged with influenza virus A/Vietnam/1194/04 (clade 1) or A/Indonesia/5/05 (clade 2.1) to assess the level of protective immunity., Results: Immunization with MVA-HA-VN/04 induced (cross-reactive) antibodies and prevented virus replication in the upper and lower respiratory tract and the development of severe necrotizing bronchointerstitial pneumonia., Conclusion: Therefore, MVA-HA-VN/04 is a promising vaccine candidate for the induction of protective immunity against highly pathogenic H5N1 avian influenza viruses in humans.

The 68k ankyrin-like protein (68k-ank) of unknown function is highly conserved among orthopoxviruses and contains ankyrin repeats and an F-box-like domain. We performed a yeast-two-hybrid screen with 68k-ank to find interacting proteins. From a human and a murine cDNA library, 99% of the interaction partners were S-phase kinase-associated protein 1a (Skp1a), a part of the SCF ubiquitin ligase complex. 68k-ank co-immunoprecipitated with components of the endogenous, mammalian SCF ubiquitin ligase. This interaction was F-box domain dependent and could also be observed in infected cells, indicating that SCF complex formation might be important for the viral life cycle.

Background: The immunomodulator AS101 [ammonium trichloro (dioxoethylene-O,O') tellurate], a nontoxic tellurium (IV) compound, has antitumoral effects which were demonstrated in several preclinical and clinical studies., Objectives: To investigate the antitumour activity of AS101 on cutaneous T-cell lymphoma (CTCL), of which mycosis fungoides (MF) is the most frequent disease variant., Methods: We used a newly established mouse xenograft model for MF to test the effect of AS101 in vivo and analysed apoptosis induction in vitro., Results: When injected intratumorally, AS101 delayed tumour growth in a dose-dependent manner. In vitro, AS101 induced a dose-dependent G2/M arrest in the CTCL cell lines Hut78 and MyLa. Moreover, higher concentrations of AS101 induced apoptosis in MyLa cells. Programmed cell death was associated with the loss of mitochondrial transmembrane potential and activation of caspase 9 and caspase 3. AS101 also elevated intracellular reactive oxygen species (ROS) production; the antioxidant, Mn superoxide dismutase, significantly reduced the degree of apoptosis, suggesting that ROS play a key role in apoptosis induction., Conclusions: These findings indicate that AS101 may be a promising antitumour drug for CTCL.

Vaccinia virus (VACV) evolved several strategies to evade antiviral cellular defence. The vaccinia virus E3 protein for example binds and sequesters double stranded RNA (dsRNA) and counteracts interferon action. We were interested to find out whether and to what extend E3 interferes with RNA silencing mediated by short interfering RNA (siRNA) in mammalian cells. We could show that the expression of a VACV-encoded marker gene can be efficiently inhibited by siRNA independently of the presence of the E3 protein. In addition, expression of E3 had no impact on RNA polymerase III promoter-derived shRNA-induced silencing of a cellular gene in human cells. Both VACV early and late gene expression could be inhibited by siRNA. Furthermore, downregulation of the expression of the E3L gene itself by siRNA in VACV infected cells produced the previously described phenotype of a knock-out virus, which illustrates the power of siRNA for vaccinia virus gene function analysis.

Since 2003, the number of human cases of infections with highly pathogenic avian influenza viruses of the H5N1 subtype is still increasing, and, therefore, the development of safe and effective vaccines is considered a priority. However, the global production capacity of conventional vaccines is limited and insufficient for a worldwide vaccination campaign. In the present study, an alternative H5N1 vaccine candidate based on the replication-deficient modified vaccinia virus Ankara (MVA) was evaluated. C57BL/6J mice were immunized twice with MVA expressing the hemagglutinin (HA) gene from influenza virus A/Hongkong/156/97 (MVA-HA-HK/97) or A/Vietnam/1194/04 (MVA-HA-VN/04). Subsequently, recombinant MVA-induced protective immunity was assessed after challenge infection with 3 antigenically distinct strains of H5N1 influenza viruses: A/Hongkong/156/97, A/Vietnam/1194/04, and A/Indonesia/5/05. Our data suggest that recombinant MVA expressing the HA of influenza virus A/Vietnam/1194/04 is a promising alternative vaccine candidate that could be used for the induction of protective immunity against various H5N1 influenza strains.

Background: The APOBEC3G protein represents a novel innate defense mechanism against retroviral infection. It facilitates the deamination of the cytosine residues in the single stranded cDNA intermediate during early steps of retroviral infection. Most poxvirus genomes are relatively A/T-rich, which may indicate APOBEC3G-induced mutational pressure. In addition, poxviruses replicate exclusively in the cytoplasm where APOBEC3G is located. It was therefore tempting to analyze whether vaccinia virus replication is affected by APOBEC3G., Results: The replication of vaccinia virus, a prototype poxvirus, was not, however, inhibited in APOBEC3G-expressing cells, nor did other members of the APOBEC3 family alter vaccinia virus replication. HIV counteracts APOBEC3G by inducing its degradation. However, Western blot analysis showed that the levels of APOBEC3G protein were not affected by vaccinia virus infection., Conclusion: The data indicate that APOBEC3G is not a restriction factor for vaccinia virus replication nor is vaccinia virus able to degrade APOBEC3G.

Short hairpin RNA (shRNA) can be stably expressed in cells to down-modulate gene expression. While retroviral and lentiviral vectors can be used to deliver shRNAs, the restricted viral titer is the major limitation for efficient gene transfer, which is especially important for cancer gene therapy. We were interested in using replicating murine leukemia virus (MLV) to enhance the shRNA transfer. Although stem loop structures could potentially interfere with the retroviral life cycle, we were able to demonstrate that the insertion of shRNA expression cassettes into MLV did not interfere significantly with viral fitness. The virus was genetically stable and able to silence target gene expression. Our results show that replicating MLVs are excellent tools for delivering shRNAs efficiently throughout the culture and have the potential to be used for gene function elucidation or even for cancer gene therapy.