Vaccinia virus produces late mRNAs by discontinuous synthesis.

We describe the unusual structure of a vaccinia virus late mRNA. In these molecules, the protein-coding sequences of a major late structural polypeptide are preceded by long leader RNAs, which in some cases are thousands of nucleotides long. These sequences map to different regions of the viral genome and in one instance are separated from the late gene by more than 100 kb of DNA. Moreover, the leader sequences map either upstream or downstream of the late gene, are transcribed from either DNA strand, and are fused to the late gene coding sequence via a poly(A) stretch. This demonstrates that vaccinia virus produces late mRNAs by tagging the protein-coding sequences onto the 3' end of other RNAs.

Biosafety of Recombinant Adeno-associated Virus Vectors.

It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression.

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.

Rectal and vaginal immunization of mice with human papillomavirus L1 virus-like particles.

Human papillomavirus (HPV) vaccines based on L1 virus-like particle (VLP) can prevent genital HPV infection and associated lesions after three intramuscular injections. Needle-free administration might facilitate vaccine implementation, especially in developing countries. Here we have investigated rectal and vaginal administration of HPV16 L1 VLPs in mice and their ability to induce anti-VLP and HPV16-neutralizing antibodies in serum and in genital, rectal and oral secretions. Rectal and vaginal immunizations were not effective in the absence of adjuvant. Cholera toxin was able to enhance systemic and mucosal anti-VLPs responses after rectal immunization, but not after vaginal immunization. Rectal immunization with Resiquimod and to a lesser extent Imiquimod, but not monophosphoryl lipid A, induced anti-HPV16 VLP antibodies in serum and secretions. Vaginal immunization was immunogenic only if administered in mice treated with nonoxynol-9, a disrupter of the cervico-vaginal epithelium. Our findings show that rectal and vaginal administration of VLPs can induce significant HPV16-neutralizing antibody levels in secretions, despite the fact that low titers are induced in serum. Imidazoquinolines, largely used to treat genital and anal warts, and nonoxonol-9, used as genital microbicide/spermicide were identified as adjuvants that could be safely used by the rectal or vaginal route, respectively.

ViralZone: a knowledge resource to understand virus diversity.

The molecular diversity of viruses complicates the interpretation of viral genomic and proteomic data. To make sense of viral gene functions, investigators must be familiar with the virus host range, replication cycle and virion structure. Our aim is to provide a comprehensive resource bridging together textbook knowledge with genomic and proteomic sequences. ViralZone web resource (www.expasy.org/viralzone/) provides fact sheets on all known virus families/genera with easy access to sequence data. A selection of reference strains (RefStrain) provides annotated standards to circumvent the exponential increase of virus sequences. Moreover ViralZone offers a complete set of detailed and accurate virion pictures.

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Summary.  Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

A secreted high-affinity inhibitor of human TNF from Tanapox virus.

A class of secreted poxvirus tumor necrosis factor (TNF)-binding proteins has been isolated from Tanapox-infected cell supernatants. The inhibitor bound to a TNF-affinity column and was identified as the product of the 2L gene. Sequence analysis of 2L family members from other yatapoxviruses and swinepox virus yielded no sequence homology to any known cellular gene. The expressed Tanapox virus 2L protein bound to human TNF with high affinity (K(d) = 43 pM) and exhibits an unusually slow off-rate. However, 2L is unable to bind to a wide range of human TNF family members. The 2L protein can inhibit human TNF from binding to TNF receptors I and II as well as block TNF-induced cytolysis. Thus, Tanapox virus 2L represents an inhibitor of human TNF and offers a unique strategy with which to modulate TNF activity.

Immune response to mouse mammary tumour virus.

Superantigens of mouse mammary tumor virus induce a strong cognate interaction between T cells and B cells. In addition to amplifying the virus-infected B-cell pool, this superantigen-driven interaction leads to the differentiation of virus-specific B cells into plasma cells. Successful interaction between T cells and B cells is required for completion of the viral life cycle.

Importance of IL28B gene polymorphisms in hepatitis C virus genotype 2 and 3 infected patients.

BACKGROUND & AIMS: Genetic variation in the interleukin 28B (IL28B) gene has been associated with the response to interferon-alfa/ribavirin therapy in hepatitis C virus (HCV) genotype 1-infected patients. The importance of three IL28B single nucleotide polymorphisms (rs8099917, rs12980275 and rs12979860) for HCV genotype 2/3-infected patients is unknown. METHODS: In patients with chronic hepatitis C genotype 2/3 (n=267), IL28B host genotypes (rs8099917, rs12980275 and rs12979860) were analyzed for associations with sustained virologic response (SVR) to antiviral therapy with (pegylated) interferon-alfa and ribavirin and with respect to epidemiological, biochemical, and virological parameters. For comparison, hepatitis C genotype 1 patients (n=378) and healthy controls (n=200) were included. RESULTS: The rs12979860 CC genotype, lower age, and genotype 2 were significantly associated with SVR in HCV genotype 2/3-infected patients (p=0.01, p=0.03 and p=0.03, respectively). No association was observed for rs8099917 and rs12980275. In addition, an SVR in patients with rapid virologic response (RVR) was associated with the rs12979860 CC genotype (p=0.05), while for non-RVR no association was found. Furthermore, a significant association with a higher baseline viral load was observed for all three IL28B genotypes in genotype 1/2/3-infected patients. Finally, increasing frequencies of the rs12979860 CC genotypes were observed in genotype 1- (33.9%), genotype 3- (38.9%), and genotype 2-infected (51.9%) patients in comparison with healthy controls (49.0%) (p<0.01). CONCLUSIONS: In genotype 2/3-infected patients, rs12979860 was significantly associated with SVR. The frequency of the rs12979860 CC genotype is lower in HCV genotype 1 vs. genotype 2/3 patients. All major IL28B genotypes are associated with HCV-RNA concentration.

Canine distemper virus : persistence and pathology in the central nervous system

Résumé : Le virus de la maladie de Carré (en anglais: canine distemper virus, CDV) qui est pathogène pour les chiens et autres carnivores, est très semblable au virus de la rougeole humaine (en anglais MV). Ces deux virus font partie du genre des Morbillivirus qui appartient à la famille des Paramyxoviridae. Ils induisent des complications dans le système nerveux central (SNC). Au stade précoce et aigu de l'infection du SNC, le CDV induit une démyélinisation (1). Ce stade évolue dans certains cas vers une infection chronique avec progression de la démyélinisation. Pendant le stade précoce, qui suit en général de trois semaines les premiers symptômes, le processus de démyélinisation est associé à la réplication du virus et n'est pas considéré comme inflammatoire (1). Par contre, au stade chronique, la progression des plaques de démyélinisation semble être plutôt liée à des processus immunogènes caractéristiques (2), retrouvés également dans la sclérose en plaques (SEP) chez les humains. Pour cette raison, le CDV est considéré comme un modèle pour la SEP humaine et aussi pour l'étude des maladies et complications induites par les Morbillivirus en général (3). Dans notre laboratoire, nous avons utilisé la souche A75/17-CDV, qui est considérée comme le modèle des souches neurovirulentes de CDV. Nous avons cherché en premier lieu à établir un système robuste pour infecter des cultures neuronales avec le CDV. Nous avons choisi les cultures primaires de l'hippocampe du nouveau-né de rat (4), que nous avons ensuite infecté avec une version modifiée du A75/17, appelée rgA75/17-V (5). Dans ces cultures, nous avons prouvé que le CDV infecte des neurones et des astrocytes. Malgré une infection qui se diffuse lentement entre les cellules, cette infection cause une mort massive aussi bien des neurones infectés que non infectés. En parallèle, les astrocytes perdent leur morphologie de type étoilé pour un type polygonal. Finalment, nous avons trouvé une augmentation importante de la concentration en glutamate dans le milieu de culture, qui laisse présumer une sécrétion de glutamate par les cultures infectées (6). Nous avons ensuite étudié le mécanisme des effets cytopathiques induits par le CDV. Nous avons d'abord démontré que les glycoprotéines de surface F et H du CDV s'accumulent massivement dans le réticulum endoplasmique (RE). Cette accumulation déclenche un stress du RE, qui est caractérisé par une forte expression du facteur de transcription proapoptotique CHOP/GADD 153 et de le la calreticuline (CRT). La CRT est une protéine chaperonne localisée dans le RE et impliquée dans l'homéostasie du calcium (Ca2+) et dans le repliement des protéines. En transfectant des cellules de Vero avec des plasmides codant pour plusieurs mutants de la glycoprotéine F de CDV, nous avons démontré une corrélation entre l'accumulation des protéines virales dans le RE et l'augmentation de l'expression de CRT, le stress du RE et la perte de l'homéostasie du Ca2+. Nous avons obtenu des résultats semblables avec des cultures de cellules primaires de cerveau de rat. Ces résultats suggèrent que la CRT joue un rôle crucial dans les phénomènes neurodégénératifs pendant l'infection du SNC, notamment par le relazgage du glutamate via le Ca2+. De manière intéressante, nous démontrons également que l'infection de CDV induit une fragmentation atypique de la CRT. Cette fragmentation induit une re-localisation et une exposition sélective de fragments amino-terminaux de la CRT, connus pour êtres fortement immunogènes à la surface des cellules infectées et non infectées. A partir de ce résultat et des résultats précédents, nous proposons le mécanisme suivant: après l'infection par le CDV, la rétention dans le RE des protéines F et H provoque un stress du RE et une perte de l'homéostasie du Ca2+. Ceci induit la libération du glutamate, qui cause une dégénération rapide du SNC (sur plusieurs jours ou semaines) correspondant à la phase aiguë de la maladie chez le chien. En revanche, les fragments amino-terminaux de la CRT libérés à la surface des cellules infectées peuvent avoir un rôle important dans l'établissement d'une démyélinisation d'origine immunogène, typique de la phase chronique de l'infection de CDV. Summary : The dog pathogen canine distemper virus (CDV), closely related to the human pathogen measles virus (MV), belongs to the Morbillivirus genus of the Paramyxoviridae family. Both CDV and NIV induce complications in the central nervous system (CNS). In the acute early stage of the infection in CNS, the CDV infection induces demyelination. This stage is sometimes followed by a late persistent stage of infection with a progression of the demyelinating lesions (1). The acute early stage occurs around three weeks after the infection and demyelinating processes are associated with active virus replication and are not associated to inflammation (1). In contrast during late persistent stage, the demyelination plaque progression seems to be mainly due to an immunopathological process (2), which characteristics are shared in many aspects with the human disease multiple sclerosis (MS). For these reasons, CDV is considered as a model for human multiple sclerosis, as well as for the study of Morbillivirus-mediated pathogenesis (3). In our laboratory, we used the A75/17-CDV strain that is considered to be the prototype of neurovirulent CDV strain. We first sought to establish a well characterized and robust model for CDV infection of a neuronal culture. We chose primary cultures from newborn rat hippocampes (4) that we infected with a modified version of A75/17, called rgA75/17-V (5). In these cultures, we showed that CDV infects both neurons and astrocytes. While the infection spreads only slowly to neighbouring cells, it causes a massive death of neurons, which includes also non-infected neurons. In parallel, astrocytes undergo morphological changes from the stellate type to the polygonal type. The pharmacological blocking of the glutamate receptors revealed an implication of glutamatergic signalling in the virus-mediated cytopathic effect. Finally, we found a drastic increase concentration of glutamate in the culture medium, suggesting that glutamate was released from the cultured cells (6). We further studied the mechanism of the CDV-induced cytopathic effects. We first demonstrated that the CDV surface glycoprotein F and H markedly accumulate in the endoplasmic reticulum (ER). This accumulation triggers an ER stress, which is characterized by increased expression of the proapoptotic transcription factor CHOP/GADD 153 and calreticulin (CRT). CRT is an ER resident chaperon involved in the Ca2+ homeostasis and in the response to misfolded proteins. Transfections of Vero cells with plasmids encoding various CDV glycoprotein mutants reveal a correlation between accumulation of viral proteins in the ER, CRT overexpression, ER stress and alteration of ER Ca2+ homeostasis. Importantly, similar results are also obtained in primary cell cultures from rat brain. These results suggest that CRT plays a crucial role in CNS infection, particularly due to CRT involvement in Ca2+ mediated glutamate releases, and subsequent neurodegenerative disorders. Very intriguingly, we also demonstrated that CDV infection induces an atypical CRT fragmentation, with relocalisation and selective exposure of the highly immunogenic CRT N-terminal fragments at the surface of infected and neighbouring non-infected cells. Altogether our results combined with previous findings suggest the following scenario. After CDV infection, F and H retention alter Ca2+ homeostasis, and induce glutamate release, which in turn causes rapid CNS degeneration (within days or a week) corresponding to the acute phase of the disease in dogs. In contrast, the CRT N-terminal fragments released at the surface of infected cells may rather have an important role in the establishment of the autoimmune demyelination in the late stage of CDV infection.

Superantigen-reactive CD4+ T cells are required to stimulate B cells after infection with mouse mammary tumor virus.

Superantigens are defined by their ability to stimulate a large fraction of T cells via interaction with the T cell receptor (TCR) V beta domain. Endogenous superantigens, classically termed minor lymphocyte-stimulating (Mls) antigens, were recently identified as products of open reading frames (ORF) in integrated proviral copies of mouse mammary tumor virus (MMTV). We have described an infectious MMTV homologue of the classical endogenous superantigen Mls-1a (Mtv-7). The ORF molecules of both the endogenous Mtv-7 and the infectious MMTV(SW) interact with T cells expressing the TCR V beta 6, 7, 8.1, and 9 domains. Furthermore, the COOH termini of their ORF molecules, thought to confer TCR specificity, are very similar. Since successful transport of MMTV from the site of infection in the gut to the mammary gland depends on a functional immune system, we were interested in determining the early events after and requirements for MMTV infection. We show that MMTV(SW) infection induces a massive response of V beta 6+ CDC4+ T cells, which interact with the viral ORF. Concomitantly, we observed a B cell response and differentiation that depends on both the presence and stimulation of the superantigen-reactive T cells. Furthermore, we show that B cells are the main target of the initial MMTV infection as judged by the presence of the reverse-transcribed viral genome and ORF transcripts. Thus, we suggest that MMTV infection of B cells leads to ORF-mediated B-T cell interaction, which maintains and possibly amplifies viral infection.

Subversion of host immune responses by viral superantigens.

Recent experiments with mouse mammary tumor virus indicate that expression of a virally encoded superantigen by B cells and its subsequent recognition by T cells are essential steps for amplification of infection and virus transmission. Preliminary results suggest that superantigens may also be expressed during retroviral infection in humans.

Hepatitis E Virus seroprevalence and chronic infections in patients with HIV, Switzerland.

We screened 735 HIV-infected patients in Switzerland with unexplained alanine aminotransferase elevation for hepatitis E virus (HEV) immunoglobulin G. Although HEV seroprevalence in this population is low (2.6%), HEV RNA can persist in patients with low CD4 cell counts. Findings suggest chronic HEV infection should be considered as a cause of persistent alanine aminotransferase elevation.

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.