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A fines de diciembre de 2019, un nuevo coronavirus (SARS-CoV-2) fue identificado como el agente causal de una nueva enfermedad respiratoria llamada COVID-19 por la OMS Sus sintomas incluyen fiebre, tos seca y dificultad respiratoria Estos sintomas en general son leves, aunque, pueden ser fatales en adultos mayores y pacientes con comorbilidades Se realizo busqueda bibliografica en Pubmed y Clinical Key donde se seleccionaron 22 articulos de acuerdo con los criterios de inclusion SARS-CoV-2 pertenece al genero de los Betacoronavirus y tiene similitudes genomicas con SARS-CoV y MERS-CoV El virion de SARS-CoV-2 consta de una nucleocapside y de una envoltura externa compuesta por proteinas estructurales principales y accesorias Su material genetico consiste en una cadena de RNA monocatenario de polaridad positiva, en el que, se codifican proteinas importantes para su transcripcion y replicacion El mecanismo de infeccion de SARS-CoV-2 comienza con la union del virion a un receptor (ACE2) de la celula huesped y su posterior entrada por endocitosis El genoma RNA viral se libera al citoplasma donde se transcriben y se traducen las proteinas necesarias para la produccion de las proteinas estructurales y para la replicacion de su material genetico Posteriormente, el RNA replicado se asocia con la nucleocapside y se ensambla junto con las proteinas estructurales para conformar las particulas viricas que seran liberadas de la celula infectada El sistema inmune hace frente a la infeccion viral mediante el reconocimiento de patrones moleculares asociados a patogenos (PAMPs) por parte de la inmunidad innata y por la accion de los linfocitos T y B por parte de la inmunidad humoral El conocimiento de las bases geneticas y moleculares de SARS-CoV-2 permite visualizar la posibilidad de establecer tratamientos farmacologicos o desarrollo de vacunas para controlar y disminuir los efectos patogenicos de la enfermedad In late December 2019, a new coronavirus (SARS-CoV-2) was identified as a causative agent of a new respiratory disease called COVID-19 by WHO Its symptoms include fever, dry cough, and shortness of breath Generally, these symptoms are mild, although, can be fatal in older adults and patients with comorbidities A bibliographic search was carried out in Pubmed and Clinical Key 22 articles were selected according to inclusion criteria SARS-CoV-2 belongs to the genus of Betacoronaviruses and has genomic similarities to SARS-CoV and MERS-CoV SARS-CoV-2 virion is made up of a nucleocapsid and external envelope composed of main structural and accesory proteins Its genetic is a positive sense single stranded RNA in which important proteins are encoded for their transcription and replication The mechanism of SARS-CoV-2 infection begins with the binding of the virion to (ACE2) receptor of the host cell and subsequent entry by endocytosis This RNA genome is released into cytoplasm and the necessary proteins for the production of structural proteins and the replication of genetic material are transcribed and translated Then, the replicated RNA associates with the nucleocapsid and assembles together with the structural proteins to form the viral particles that will be released from the infected cell The immune system faces viral infection through the recognition of molecular patterns associated with pathogens (PAMPs) by innate immunity and the action of T cells and B cells by humoral immunity Knowledge of the genetic and molecular basis of SARS-CoV-2 allows us to visualize the possibility of establishing pharmacological or vaccine treatments to control and reduce the pathogenic effects of the disease

Oncolytic viruses are designed to replicate in and kill cancer cells, and have shown tremendous promise in preclinical and clinical studies. Indeed, several oncolytic viruses are available to patients in a number of different countries around the world. However, most oncolytic viruses show a poor ability to spread throughout the tumor mass, frequently leading to only a partial response and regrowth of the tumor. One approach to improve spread of the viral effect throughout the tumor mass is to arm the oncolytic virus with a fusogenic protein. In this manner, a single infected cell can fuse with many adjacent uninfected cells, essentially amplifying the anti-tumor effects. In this review, we discuss the development and use of fusogenic proteins to enhance the efficacy of human adenovirus-based vectors for cancer therapy.

Mycoplasma contamination of cell line cultures is a common, yet often undetected problem in research laboratories. Many of the existing techniques to detect mycoplasma contamination of cultured cells are time-consuming, expensive, and have significant drawbacks. Here, we describe a mycoplasma detection system that is useful for detecting multiple species of mycoplasma in infected cell lines. The system contains three dye-labeled detection aptamers that can specifically bind to mycoplasma-infected cells and a dye-labeled control aptamer that minimally binds to cells. With this system, mycoplasma-contaminated cells can be detected within 30 min by using a flow cytometer, fluorescence microscope, or microplate reader. Further, this system may be used to detect mycoplasma-contaminated culture medium. This study presents an novel mycoplasma detection model that is simple, rapid, inexpensive, and sensitive.

PhageFISH uses the power of fluorescence in situ hybridization to monitor intracellular phage infections at single cell level. It combines host cell identification via rRNA probes and phage identification via phage-specific gene probes, allowing for the quantification of the infected cell fraction and the discrimination between infection stages. This book chapter covers all aspects of the procedure, from phage probe design and synthesis, to the phageFISH protocol itself, to microscopy and image analysis.

Many investigations of the replication and pathogenesis of human T-cell leukemia virus type 1 (HTLV-1) employ chronically infected cell lines, cell lines stabilized from primary adult T-cell leukemia cells, and noninfected T-cell lines. The validity of data obtained from such studies depends on the unambiguous identification of each cell line, which can be performed by short-tandem-repeat (STR) profiling (DNA fingerprinting). While kit-based profiling represents the standard method for cell line authentication, not all labs have ready access to the required capillary electrophoresis equipment, and the costs of such tests can become substantial, especially if the cell lines are to be tested frequently. We analyzed DNA from a panel of HTLV-1-infected cell lines and noninfected T-cell lines using a commercial STR kit and then analyzed the same DNA for individual STR markers followed by nondenaturing polyacrylamide gel electrophoresis. This simplified method should facilitate routine confirmation of cell line identity in diverse laboratory settings.

We established abnormal isoform of prion protein (PrPSc)-specific double immunostaining using mAb 132, which recognizes aa 119–127 of the PrP molecule, and novel PrPSc-specific mAb 8D5, which recognizes the N-terminal region of the PrP molecule. Using the PrPSc-specific double immunostaining, we analysed PrPSc in immortalized neuronal cell lines and primary cerebral-neuronal cultures infected with prions. The PrPSc-specific double immunostaining showed the existence of PrPSc positive for both mAbs 132 and 8D5, as well as those positive only for either mAb 132 or mAb 8D5. This indicated that double immunostaining detects a greater number of PrPSc species than single immunostaining. Double immunostaining revealed cell-type-dependent differences in PrPSc staining patterns. In the 22 L prion strain-infected Neuro2a (N2a)-3 cells, a subclone of N2a neuroblastoma cell line, or GT1-7, a subclone of the GT1 hypothalamic neuronal cell line, granular PrPSc stains were observed at the perinuclear regions and cytoplasm, whereas unique string-like PrPSc stains were predominantly observed on the surface of the 22 L strain-infected primary cerebral neurons. Only 14 % of PrPSc in the 22 L strain-infected N2a-3 cells were positive for mAb 8D5, indicating that most of the PrPSc in N2a-3 lack the N-terminal portion. In contrast, nearly half PrPSc detected in the 22 L strain-infected primary cerebral neurons were positive for mAb 8D5, suggesting the abundance of full-length PrPSc that possesses the N-terminal portion of PrP. Further analysis of prion-infected primary neurons using PrPSc-specific immunostaining will reveal the neuron-specific mechanism for prion propagation.

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

Zusammenfassung Infizierte Zellkulturen eignen sich gut zur Gewinnung komplementbindender Antigene fur die Herpesvirus equi 1 (Rhinopneumonitis)-Serodiagnose. Das komplementbindende Antigen kann in der erforderlichen Konzentration aus der Zellphase mit hohen Dosen infizierter Kulturen gewonnen werden. Die Kulturantigene sind den herkommlichen Antigenen aus Lebern infizierter Hamster oder Organen abortierter Pferdefeten in ihren praktischen Eigenschaften uberlegen. In den eigenen Untersuchungen wurden Antigene aus BHK-Kulturen bevorzugt, weil sie einer heterologen Zellart entstammen, in befriedigender Konzentration herstellbar sind und bei ihrer Verwendung keine unspezifischen Reaktionen befurchtet werden mussen. Die Eignung komplementbindender Rhinopneumonitisantigene, gewonnen aus primaren Schweinenieren- und BHK-Kulturen, wurde in serologischen Routineuntersuchungen an Pferdeseren gepruft. Dabei wurde festgestellt, das die Verbreitung der Rhinopneumonitisinfektion im Raume Munchen-Oberbayern noch keinen rucklaufigen Trend aufweist. Summary Complement-fixing antibodies of herpes virus equi 1 (rhino-pneumonitis virus) from cell cultures and their use in serological diagnosis Infected cell cultures are very suitable for the production of complement-fixing antibodies for the serological diagnosis of herpes virus equi 1. The antigen can be obtained in the required concentration from the cell phase with high doses of infected cultures. The culture antigens are superior to the conventional antigens prepared from the livers of infected hamsters or from the organs of aborted equine foetuses. The authors prefer antigens from BHK cultures because they come from a heterologous cell species, they are available in suitable concentration and nonspecific reactions are unlikely. The quality of complement-fixing rhino-pneumonitis antigens produced from primary pig kidney and BHK cultures was tested in routine serological studies on horse sera. The results showed that there is still no tendency for regression in the spread of rhino-pneumonitis infection in the Munich-Upper Bavaria area. Resume Antigenes du virus herpes equi 1 (rhinopneumonnitisvirus), fixant le complement, obtenus en cultures de cellules; leur application dans le diagnostic serologique Des cultures de cellules infectees conviennent fort bien a la preparation des antigenes fixant le complement pour le diagnostic du virus herpetique equi 1 (rhinopneumonitis). On obtient l'antigene fixant le complement, a la concentration requise, a partir de la phase cellulaire de cultures infectees a fortes doses. Les antigenes de cultures sont superieurs aux antigenes conventionnels prepares a partir de foies de hamsters infectes ou d'organes de foetus equins avortes, grâce a leurs proprietes pratiques. Dans nos recherches personelles, nous preferons les antigenes cultures BHK, parce qu'ils proviennent d'une espece cellulaire heterologue, qu'on en obtient des concentrations satisfaisantes et que utilisation n'entraine pas de reactions non specifiques. La qualite des antigenes de la rhinopneumonie, fixant le complement, prepares a partir de cultures primaires de reins de porcs et de cultures BHK, est testee dans des exames serologiques de routine sur des serums equins. On trouve que la propagation de l'infection de la rhinopneumonie ne manifeste encore aucune tendance a la regression dans la region de Munich — Haut Baviere. Resumen Antigenos fijadores del complemento del virus herpetico equi 1 (virus de la rinopneumonitis) a partir de cultivos celulares y su aplicacion en el diagnostico serologico Los cultivos celulares infectados resultan muy apropiados para obtener antigenos fijadores del complemento destinados el serodiagnostico del virus herpetico equi 1 (rinopneumonitis). El antigeno fijador del complemento se puede conseguir en la concentracion precisa de la fase celular de cultivos infectados con dosis elevadas. Los antigenos celulares son superiores en sus propiedades practicas a los antigenos inveterados de higados de hamsters infectados o de organos de fetos abortados de yegua. En los estudios propios se prefirieron los antigenos de cultivos BHK (rinones de hamsters bebes), ya que proceden de una especie celular heterologa, se pueden elaborar en concentracion satisfactoria y no se precisan temer reacciones inespecificas con su empleo. La idoneidad de los antigenos fijadores del complemento de la rinopneumonitis, obtenidos a partir de cultivos primarios de rinones porcinos y BHK, se contrasto en pesquisas serologicas de rutina con sueros sanguineos equinos. De esta forma se pudo comprobar que la difusion de la infeccion rinopneumonitica aun no presenta ninguna tendencia retrograda en el territorio de Munich-Alta Baviera.

In this study, the infection cycle of bacteriophage Qbeta was investigated. Adsorption of bacteriophage Qbeta to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4x10(-10) ml/cells/min. In infected cells, approximately 130 molecules of beta-subunit and 2x10(5) molecules of coat protein were translated in 15 min. Replication of Qbeta RNA proceeded in 2 steps-an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5x10(5) molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.

The existing models of hepatitis B virus (HBV) infection dynamics are based on the assumption that the populations of viruses and cells are uniformly mixed. However, the real virus infection system is actually not homogeneous and some spatial factors might play a nontrivial role in governing the development of HBV infection and its outcome. For instance, the localized populations of dead cells might adversely affect the spread of infection. To consider this kind of inhomogeneous feature, a simple 2D (dimensional) probability Cellular Automaton model was introduced to study the dynamic process of HBV infection. The model took into account the existence of different types of HBV infectious and non-infectious particles. The simulation results thus obtained showed that the Cellular Automaton model could successfully account for some important features of the disease, such as its wide variety in manifestation and its age dependency. Meanwhile, the effects of the model's parameters on the dynamical process of the infection were also investigated. It is anticipated that the Cellular Automaton model may be extended to serve as a useful vehicle for studying, among many other complicated dynamic biological systems, various persistent infections with replicating parasites.

Retroviral assembly proceeds through a series of concerted events that lead to the formation and release of infectious virion particles from the infected cell. Upon translation, structural proteins are targeted to the plasma membrane where they accumulate. There, the nascent particle forces the plasma membrane to form a bud, which pinches off releasing the virion particle from the cell. In this review we describe the molecular mechanisms now known to be behind the process of virion assembly. In particular, we focus on the human immunodeficiency virus type 1, the prototype member of the lentivirus subfamily of the Retroviridae.

The herpes simplex virus 1 capsid is formed in the infected cell nucleus by way of a spherical, less robust intermediate called the procapsid. Procapsid assembly requires the capsid shell proteins (VP5, VP19C, and VP23) plus the scaffolding protein, pre-VP22a, a major component of the procapsid that is not present in the mature virion. Pre-VP22a is lost as DNA is packaged and the procapsid is transformed into the mature, icosahedral capsid. We have employed a cell-free assembly system to examine the role of the scaffolding protein in procapsid formation. While other reaction components (VP5, VP19C, and VP23) were held constant, the pre-VP22a concentration was varied, and the resulting procapsids were analyzed by electron microscopy and SDS-polyacrylamide gel electrophoresis. The results demonstrated that while standard-sized (T = 16) procapsids with a measured diameter of approximately 100 nm were formed above a threshold pre-VP22a concentration, at lower concentrations procapsids were smaller. The measured diameter was approximately 78 nm and the predicted triangulation number was 9. No procapsids larger than the standard size or smaller than 78-nm procapsids were observed in appreciable numbers at any pre-VP22a concentration tested. SDS-polyacrylamide gel analyses indicated that small procapsids contained a reduced amount of scaffolding protein compared to the standard 100-nm form. The observations indicate that the scaffolding protein concentration affects the structure of nascent procapsids with a minimum amount required for assembly of procapsids with the standard radius of curvature and scaffolding protein content.

L'apoptose des lymphocytes T joue un role essentiel dans les processus de developpement et de maturation du systeme immunitaire. En effet, la mort par apoptose est impliquee dans l'etape de maturation intrathymique qui conduit a la deletion clonale de lymphocytes T auroreactifs et a l'etablissement de la tolerance au soi, elle joue un role important dans l'homeostase des lymphocytes T matures repondeurs a une stimulation antigenique, en limitant leur expansion clonale, et elle contribue a l'elimination de cellules infectees par un virus tels que le VIH (virus de l'immunodeficience humaine). Le VIH a la particularite de s'attaquer a une cellule clef du systeme immunitaire, le lymphocyte T auxiliaire, qui exprime a sa surface la molecule CD4, recepteur specifique du VIH. Au travers des cytokines qu'ils synthetisent, les lymphocytes T CD4 aident les effecteurs de l'immunite naturelle, tels que les cellules NK (natural killer), les lymphocytes T γδ et les macrophages, a detruire les cibles infectees par le VIH. Par ailleurs, ils sont essentiels a l'activation et la maturation des lymphocytes B en cellules productrices d'anticorps, ils sont requis pour la differenciation des lymphocytes T CD8 en cellule cytotoxique (CTL) specifique du VIH, et ils representent une source de chimiokines, facteurs inhibiteurs de l'entree du VIH dans la cible T CD4 [32]. En consequence, la disparition progressive de ces lymphocytes T auxiliaires, dependante du VIH, conduit a un defaut de controle par le systeme immunitaire de la replication virale, a la destruction des organes lymphoides ou a lieu la reponse immunitaire, et a l'apparition du syndrome d'immunodeficience acquise (sida), caracterise par le developpement d'infections opportunistes severes pouvant conduire au deces du patient. Les mecanismes responsables de la disparition des lymphocytes T CD4 au cours de l'infection par le VIH sont complexes, et ils ne sont que partiellement elucides. La destruction des lymphocytes T CD4 peut etre la consequence directe de l'infection virale, ou indirecte au travers de l'activation par les proteines du VIH de certaines voies pro-apoptotiques dans des lymphocytes non infectes. Par ailleurs, le systeme immunitaire peut aussi contribuer a cette destruction lymphocytaire, puisque les cellules infectees peuvent etre detruites par les CTL ou par un mecanisme de cytotoxicite dependante des anticorps (ADCC).

A long trail of evidence indicates that the formation of PrPSc or its accumulation causes the neuronal dysfunction and clinical features of prion diseases. The results of our current line of studies argue that the main neuropathological and clinical features of prion diseases are explained by altered ion channel function secondary to decreased plasma membrane fluidity. This kind of mechanism has the potential to functionally disconnect neuronal networks and cause neuronal vacuolation. Our laboratory is currently focusing its investigations on pathogenic mechanisms that have the potential to link the formation of PrPSc with plasma membrane abnormalities in prion diseases. In summary, the first hypothesis suggests that the conversion of PrPC to PrPSc affects plasma membrane fluidity directly, which secondarily alters the properties and functions of its components. In contrast, the second hypothesis argues that PrPSc accumulation alters the ability of chaperones to correctly fold plasma-membrane proteins during their synthesis, which directly affects the properties of nascent proteins and secondarily affects membrane fluidity. Our current investigations are attempting to determine which of these mechanisms are plausible and, then, which is primary.

Reducing agents such as glutathione (GSH), glutathione ester (GSE), and N-acetylcysteine (NAC) have been shown to suppress the induction of HIV expression in chronically infected cells stimulated by cytokines. We present data which show the effects of the organic thiophosphate WR-151327 on the expression of latent HIV in U1 cells. The chronically infected promonocytic cell line U1 constitutively expresses low levels of HIV that can be increased by 13-phorbol 12-myristate acetate (PMA), tumor necrosis factor alpha (TNF-alpha), and granulocyte/monocyte colony-stimulating factor (GM-CSF). WR-151327 suppressed, in dose-dependent fashion, the reverse transcriptase (RT) activity induced by TNF-alpha, GM-CSF, and PMA. The maximal decrease in RT activity was 70, 80, and 50%, respectively. Pretreatment with WR-151327 also suppressed the induction of total HIV protein synthesis, as shown by Western blot analysis. In addition, WR-151327 suppressed HIV-LTR-CAT activity in transfected human rhabdomyosarcoma cells (RD). Suppression of HIV expression by WR-151327 was observed in the absence of a cytotoxic or cytostatic effect. Incubation of WR-151327 with human recombinant TNF-alpha for 6 hr at 37 degrees C did not alter the capacity of TNF-alpha to induce the expression of HIV. Our observations further support the hypothesis that reducing agents are important in the control of HIV replication and that the clinical evaluation of WR-151327 may be indicated.

A rapid, high-capacity assay for evaluating the potency of anti-HIV compounds was devised. This assay measures cell associated viral RNA levels 3 days after infection of susceptible T-cell lines grown in individual microtitre plate wells. Viral RNA was quantified by a sandwich hybridization assay, the first step of which was performed directly in crude infected cell lysates prepared in guanidinium isothio-cyanate. Levels of cell-associated viral RNA were shown to correlate with the yield of infectious virus and this correlation formed the basis of the test. Antiviral potencies of a large series of compounds tested in this RNA hybridization assay correlated closely with potency values determined by a sensitive but slower and more labour-intensive yield-reduction assay. Both laboratory strains and selected clinical isolates of HIV can be detected in this RNA hybridization assay.

The ‘Springer Index of Viruses’, edited by C.A. Tidona and G. Darai, marks the first time in which a new technique for the presentation of data on viruses, especially for virus genes and the viral proteins, is utilized. The editors have developed a method that allows for the succinct depiction of a copious amount of referenced data on 241 virus genera, and on 2196 virus species. Three hundred and forty one scientists collaborated on the Index of Viruses.

Summary The sequence of the MHV-A 59 non-structural gene 4 (ns 4) reveals two open reading frames. The upstream ORF potentially encodes a 19 amino acid (2.2 kDa) polypeptide and the downstream ORF potentially encodes a 106 amino acid (11.7 kDa) polypeptide. This is in contrast to MHV-JHM gene 4 which expresses a 15 kDa protein. Cell free translation of a synthetic mRNA containing both ORFs of MHV-A 59 ns 4 results in the synthesis of a 2.2 kDa poylpeptide; the predicted 11.7 kDa product of the MHV-A 59 downstream ORF is not detected during cell free translation nor in infected cells. These results add to the recent data suggesting that expression of some of the ns proteins of MHV is not necessary for efficient growth in tissue culture.

L’activite des principes actifs habituellement utilises au cours des leishmanioses a pu etre amelioree, en les incorporant a un vecteur.Nous presentons un modele in vitro permettant l’etude des interactions leishmanies-nanoparticules chargees ou non chargees. Ce travail a ete realise avec deux types de nanoparticules : des nanospheres de polyisoalkylcyanoacrylate, vecteur particulaire, biodegradable et des nanospheres de methacrylate non chargees et chargees de pentamidine.Le modele associe la lignee mono-histiocytaire U397 et des formes amastigotes de Leishmania major. La methodologie a consiste a mettre en contact, des concentrations de l’ordre de 5 a 50 µg/ml pour les polyisoalkylcyanoacrylates et de 50 a 200 µg/ml pour les polymeres de methacrylate vis-a-vis de 3.105 cellules infectees/ml ; l’efficacite des nanoparticules est evaluee 24 h plus tard en microscopie optique par mesure de l’index de l’infestation residuelle.Nous presentons ici les resultats des essais aux concentrations pour lesquelles nous obtenons une alteration ou une disparition des leishmanies dans les vacuoles d’endocytose.La validite et l’interet de ces resultats sont discutes.

Although potent combination therapy is usually able to suppress plasma viral loads in HIV-1 patients to below the detection limit of conventional clinical assays, a low level of viremia frequently can be detected in plasma by more sensitive assays. Additionally, many patients experience transient episodes of viremia above the detection limit, termed viral blips, even after being on highly suppressive therapy for many years. An obstacle to viral eradication is the persistence of a latent reservoir for HIV-1 in resting memory CD4+ T cells. The mechanisms underlying low viral load persistence, slow decay of the latent reservoir, and intermittent viral blips are not fully characterized. The quantitative contributions of residual viral replication to viral and the latent reservoir persistence remain unclear. In this paper, we probe these issues by developing a mathematical model that considers latently infected cell activation in response to stochastic antigenic stimulation. We demonstrate that programmed expansion and contraction of latently infected cells upon immune activation can generate both low-level persistent viremia and intermittent viral blips. Also, a small fraction of activated T cells revert to latency, providing a potential to replenish the latent reservoir. By this means, occasional activation of latently infected cells can explain the variable decay characteristics of the latent reservoir observed in different clinical studies. Finally, we propose a phenomenological model that includes a logistic term representing homeostatic proliferation of latently infected cells. The model is simple but can robustly generate the multiphasic viral decline seen after initiation of therapy, as well as low-level persistent viremia and intermittent HIV-1 blips. Using these models, we provide a quantitative and integrated prospective into the long-term dynamics of HIV-1 and the latent reservoir in the setting of potent antiretroviral therapy., Author Summary Current combination therapy can suppress viral loads in HIV-1-infected individuals to below the detection limit of standard commercial assays. However, it cannot eradicate the virus from patients. HIV-1 can generally be identified in resting memory CD4+ T cells and persists in patients on potent treatment for a long time. These latently infected cells decay slowly, but can produce new virions when activated by relevant antigens. Many patients experience transient episodes of viremia, or blips, even though they have “undetectable” plasma viral loads for many years. Here, we develop a new mathematical model describing latently infected cell activation upon random antigenic stimulation. Using the model, we show that programmed expansion and contraction of latently infected cells upon activation can generate both low viral load persistence and viral blips. Occasional replenishment of the latent reservoir may explain the different decay kinetics of the reservoir observed in clinical practice. We also show that a model with homeostatic proliferation of latently infected cells can explain persistence of low-level virus, stability of the latent reservoir, and emergence of viral blips. These results provide novel insights into the long-term virus dynamics and could have implications for the treatment of HIV-1 infection.

A polymerase chain reaction combined with restriction enzyme analysis was developed for detection and differentiation of all 12 fowl adenovirus (FAdV) serotypes representing the five fowl adenovirus (A to E) species. For primer design, the published sequences of the hexon proteins of FAdV1, FAdV8 and FAdV9 were aligned and conserved regions in the two pedestal regions adjacent to the L1 loop region were determined. A primer pair (hexon A/hexon B) was constructed and was shown to amplify approximately 900 bp of the hexon gene including the L1 loop region. An amplification product was detected using supernatant of infected cell cultures from all FAdV1 to FAdV12 reference strains used in our study. The sequence and the restriction patterns of the hexon A/B fragments of the 12 FAdV strains were determined and compared. The successive use of four different endonucleases allowed the complete differentiation of the reference FAdV strains. Twenty-six fowl adenoviruses isolated during our routine virological diagnosis activities could all be amplified using hexon A/hexon B primers. Restriction analysis results showed that 8/26 adenovirus strains contained two different FAdV types. FAdV4, FAdV12, FAdV1, FAdV5 and FAdV6 were the most frequently isolated.

Purified poliovirus 14 S subunits are assembled into empty capsids in vitro only if their concentration exceeds a 1.6 n M threshold. This also holds true for the 14 S subunits in unpurified extracts of infected cells. Such an extract may promote the assembly of extraneous 14 S subunits, but only if it contributes enough 14 S subunits to raise their total concentration over the threshold. As a result of assembly, the concentration of 14 S subunits in an infected cell extract decreases exponentially, with a half life of 15 min at 37°. When purified 14 S subunits of serotype 1 are mixed with extracts of cells infected with type 2 or 3, chimeric empty capsids are formed, thus showing the pooling of endogenous and extraneous 14 S subunits. In conclusion, the assembly promoting activity of infected cell extracts amounts to nothing more than the supply of endogenous 14 S subunits.

The replication and polypeptide synthesis of Abadina virus, a member of the Palyam serogroup of orbiviruses, was studied. The first virus-specified proteins could be demonstrated 2 to 4 h post-infection (p.i.) by immunoprecipitation. The rate of synthesis increased rapidly until 12 h p.i. after which it remained fairly constant until 18 h p.i. when it began to decline. Host cell protein synthesis shutoff was incomplete. Twelve virus-induced polypeptides were identified in infected cell lysates, ranging in M r from 36K to 143K. Three small polypeptides (M r 15K to 20K) identified in homologous immunoprecipitation studies are also thought to be virus-specified. Nine structural proteins were identified, four being major components of the purified virion. Partial proteolysis was used to demonstrate homology between some proteins. Pulse-chase experiments provided no evidence for a precursor-product relationship between any of the Abadina virus proteins. A non-structural protein was found to be phosphorylated.

HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1alpha in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1alpha protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1alpha by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1alpha protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1alpha protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.

This method is designed to measure rates of transcription from adenoviral promoters as a function of the concentrations within infected cells of the promoter(s) of interest. The latter parameter is assessed by quantification of viral DNA by hybridization of membrane-bound DNA following purification of DNA from nuclear fractions of adenovirus-infected cells. Two alternative protocols, primer extension and quantitative reverse transcription polymerase chain reaction, are described for determination of the concentrations of viral mRNAs purified from the cytoplasmic fractions of the same infected cell samples. An alternative procedure to measure rates of transcription directly using run-on transcription in isolated nuclei is also presented.

The use of anti-PrP antibodies represents one of the most promising strategies for the treatment of prion diseases. In the present study, we screened various anti-PrP antibodies with the aim of identifying those that would block PrP(Sc) replication in prion-infected cell culture. Two antibodies, SAF34 recognizing the flexible octarepeats region on HuPrP protein, and SAF61 directed against PrP amino acid residues (144-152), not only inhibited PrP(Sc) formation in prion-infected neuroblastoma cells but also decreased the PrP(C) levels in non-infected N2a cells. In addition, treatment with both SAF34 and SAF61 antibodies decreased PrP(C) and PrP(Sc) levels in the cells synergistically. In the presence of both antibodies, our results showed that the mode of action which leads to the disappearance of PrP(Sc) in cells is directly coupled to PrP(C) degradation by reducing the half-life of the PrP(C) protein.

During the course of infection, a subset of HIV-1 proteins interacts with multiple cellular partners, sometimes in a hierarchical or sequential way. These proteins include those associated with the initial infection event, with the preparation of the cell for the replicative cycle of the virus and with the exit of new virions from the infected cell. It appears that the interactions of viral proteins with multiple cellular partners are mediated by the occurrence of ligand-induced conformational changes that direct the binding of these proteins to subsequent partners. Two of the most studied HIV-1 proteins that are known to interact with different cellular partners are gp120 and Nef. Here we discuss the interactions of these two proteins with their cellular partners and present new results indicating that the conformational changes undergone by these proteins define a novel allosteric paradigm. In the traditional view, conformational changes are thought to occur between well defined structural conformations of a protein. In gp120 and Nef, those changes involve conformations characterized by the presence of large regions devoid of stable secondary or tertiary structure. Those unstructured regions contain the binding determinants for subsequent partners and only become functionally competent by ligand-induced structuring or un-structuring of those regions. By switching binding epitopes between structured and unstructured conformations the binding affinity can be modulated by several orders of magnitude, thus effectively precluding binding against unwanted partners. A better understanding of these interactions would lead to improved strategies for inhibitor design against these viral targets.

Summary. We have previously shown that mitochondrial-aconitase binds specifically to the 3′ terminal 42 nucleotides of the Murine hepatitis virus (MHV) RNA along with three additional proteins of 70, 58 and 40 kDa to form a stable RNA-protein complex. Supershift and western blot assays have identified these three proteins as mitochondrial HSP70 (mtHSP70), HSP60, and HSP40. A series of co-immunoprecipitation assays have established that these four MHV RNA binding proteins are associated, even in the absence of MHV RNA. However, the presence of a synthetic RNA containing the sequence bound by these four proteins does increase the amount of co-precipitated protein, in particular the amount of HSP60 which is brought down with antibodies directed against HSP40 and mtHSP70. We have provided evidence for the interaction of these four proteins with the 3′ end region of MHV RNA in infected cells by a series of immunoprecipitation RT-PCR assays. We believe it is likely that MHV RNA interacts with m-aconitase prior to its import into mitochondria in cooperation with extra-mitochondrial mtHSP70, HSP60, and HSP40.

Maturation of adenovirus particles is markedly affected by proteolytic processing. The possibility for blocking the conversion of precursor structural core protein (preVII) into mature structure protein VII by officinal drugs epsilon-aminocaproic acid and ambenum has been demonstrated in Hep-2 cells infected with adenovirus. Proteolytic processing may be regarded as one of the targets for inhibiting adenovirus reproduction.

We previously examined Moloney murine leukemia virus-induced fusion-from-within (FFWI) and fusion-from-without (FFWO) of SC-1 mouse cells. FFWI and FFWO can be distinguished by their stimulation by ionophores and polycations, respectively. FFWI is caused by infected cells. Normally, fusion between an infected cell and uninfected cells (heterofusions) is described, but we have surprisingly found that the infected cells also caused homofusion between uninfected cells in their vicinity (named neighbor homofusions). It was shown that neighbor homofusions were not induced by free virus particles (by FFWO). Transfectants expressing envelope proteins only induced heterofusions, indicating that virus production is necessary for the formation of neighbor homofusions. Both plasma membrane fragments and easily removable material from the surface of infected cells were able to induce fusion with the same stimulation pattern as FFWI and neighbor homofusion. These materials, especially the latter, have properties in common with virions, and it is discussed whether immature virions are involved in the formation of the neighbor homofusions.

Spontaneous transfer of protein from either influenza virus-infected or uninfected cells to dimyristoylphosphatidylcholine (DMPC) liposomes was examined. The amount of transferred protein of liposomes incubated with influenza virus-infected cells (I-lipo) was more than that of liposomes incubated with uninfected cells (U-lipo). The ratio of the amount of spontaneous transferred protein of I-lipo to that of U-lipo increased in proportion to the diameter of liposomes except for small unilamellar vesicles.

Co-infection with several viruses is often used to achieve simultaneous expression of several proteins. From co-infections involving several viruses, the ratio of proteins synthesized in individual cells can be very variable. This is disadvantageous where proteins are needed to interact to provide a maximum yield of a complex product. Multiple-gene-expression vectors offer an alternative to co-infections. They enable reproducible ratios of products to be provided in each infected cell. Until recently, multigene-expression vectors have only been developed to make two proteins simultaneously. Here, we describe the generation of a single recombinant baculovirus synthesising up to five foreign proteins with a fixed ratio comparable to the ratio during the synthesis of native proteins.

Mitochondrially encoded H antigens are by-products of a system that has evolved in vertebrates to present peptides from intracellular pathogens on the cell surface for detection by CTLs, which can lyse the infected cell. CTL lines and clones with defined specificity against mitochondrial H antigens, which can be maintained in culture for long periods, offer a unique tool in mitochondrial genetics. Expression of polymorphic mitochondrial H antigens depends on both the presence and the activity of the corresponding mitochondrial genome, and CTLs can provide strong selection against cells displaying their cognate antigen.

The effects of herpes simplex virus type I (HSV-I) on SCE frequencies in human lymphocytes in vitro were investigated. SCE frequencies in controls (mean +/- ISD 5.2 +/- 1.6) and in cells exposed to the virus for 3 and 6 h (mean +/- ISD 5.3 +/- 1.6 and 5.8 +/- 1.6 respectively) were about the same. However, cells infected for 9 and 24 h showed a significant increase of SCE frequencies (p < 0.05).

Recent studies using biotinylated in situ hybridization (ISH) have utilized a wide range of detection protocols for the biotinylated hybrids, leading to conflicting reports in the literature regarding sensitivity. In this study we compared 11 different detection protocols for biotinylated ISH using a measles virus-specific RNA probe on formalin-fixed, paraffin-embedded central nervous system tissue infected with measles virus. Maximum sensitivity was achieved with five-step detection protocols incorporating the use of a monoclonal antibody to biotin. Single-step detection protocols were found to be insensitive, as shown by their failure to detect viral nucleic acid in infected white-matter cells. Only by increasing the number of steps in the detection protocols were these infected cells demonstrable. Unless pre-hybridization, hybridization, and detection protocols are optimized, the results obtained in pathogenicity studies using ISH could be misinterpreted, leading to false conclusions about nucleic acid distribution. This also applies to the ever-increasing use of ISH for diagnostic purposes.

Three bovine herpesvirus type 4 (BHV-4) proteins released into the culture medium of infected cells were identified, with M r values of 135K, 16K and 14.5K. Among these three proteins, two were precipitated by the monoclonal antibodies characterized in this work. One is a glycoprotein of 135K (gp8) which does not seem to be involved in BHV-4 neutralization. Moreover, this 135K glycoprotein adsorbed onto uninfected susceptible cells. The attachment of gp8 to cells was totally inhibited by the prior adsorption of unlabelled viral proteins. Moreover, anti-gp8 monoclonal antibodies were effective in inhibiting the adsorption of gp8. These results indicate that gp8 could be involved in BHV-4 attachment.

Nucleic acid hybridization methods in routine diagnosis of micro-organisms have been limited by the tedious assay procedures. We have previously described the sandwich hybridization method which allows convenient testing of biological specimens. In this paper we describe the adaptation of the solution hybridization method into the microtitre plate format using 35 S-isotope as label. Using 3-hour hybridization followed by 2-hour collection of the hybrids a sensitivity of 5 × 10 5 target DNA molecules was achieved. The method was applied for identification of human papillomaviruses in crude gynaecological specimens. A simple 1-day assay protocol was achieved with high HPV type specificity. The specificity was confirmed by testing a variety of unrelated micro-organisms, none of which gave a positive signal in the test. Results, obtained as numerical values, were easy to interpret; positive and negative samples gave clearly distinguishable signals.

The use of pre-embedding immunogold-silver staining (IGSS) in electron microscopy of cell culture monolayers has been reported by various authors and its usefulness to localize antigens has been documented. Embedding cell suspensions or cell fractions for IGSS can require time-consuming centrifugation steps. Embedding cell suspensions in agarose simplifies IGSS processing and embedding procedures.Bovine turbinate cells (BTs) were grown to confluence in 25cm3culture flasks. BTs were infected with a non-cytopathic strain (ncp) of bovine viral diarrhea virus (BVDV) while control cells were sham inoculated. At 18 h pi, cells were detached with trypsin. All following procedures were carried out at RT. The cells were fixed for 30 min in 2.0% paraformaldehyde + 0.2% glutaraldehyde in 0.1 M. phosphate buffer, pH 7.4, and rinsed 3 times in 0.1 M phosphate buffer, pH 7.4 (each centrifuged for 10 min at 300g). The cells were resuspended in 1 ml of buffer, transferred to an eppendorf tube and centrifuged for 10 min at 300g. The excess buffer was removed, 1-2 drops of warm, molten 1.0% agarose added, and the mixture centrifuged for 1 min at 300g to pellet the cells to the tip of the tube. The solidified cell-agarose mixture was removed, cut into 3pieces, and placed in phosphate buffer.

A cytopathic strain of bovine viral diarrhea virus (BVDV) was purified from infected cell culture fluids by isopycnic density-gradient centrifugation. Genomic RNA was extracted and tailed with adenine residues at the 3′ end with poly-A polymerase. Double-stranded complementary DNA (cDNA) was synthesized, using the poly-A-tailed RNA as a template and oligo-dT as a primer, and then cloned into the pUC9 plasmid. Virusspecific cDNA sequences, varying in length from 0.5 to 2.5 kilobases (kb), were obtained. One BVDV-specific sequence of cloned cDNA, 1.1 kb in length and with an internal Pst I restriction endonuclease cleavage site, was selected for use as a probe. The cloned cDNA insert was removed from the plasmid either with or without flanking plasmid sequences and labeled with 32P-nucleotides by nick translation for use as hybridization probes for BVDV. The performance of probes of smaller fragments of the insert was compared to that of the intact sequence in hybridization assays. In addition, 2 methods of specimen preparation were compared to establish optimum parameters for hybridization. The hybridization assay was 10–100 times more sensitive than infectivity assays for BVDV in infected cell cultures. Freezing of specimens reduced by lo-fold the sensitivity of hybridization for BVDV target sequences. The probes prepared from the cloned cDNA hybridized with all cytopathic and noncytopathic BVDV strains tested but not with uninfected cell cultures, cellular ribosomal RNA, bovine coronavirus, bluetongue virus, or bovine adenovirus 3. Probes prepared with native plasmid DNA did not hybridize with BVDV or uninfected cell cultures. Probes of 1.1 kb, 0.6 kb, and 0.5 kb hybridized with equal sensitivity to target sequences in the dot blot system. The presence of flanking plasmid sequences did not improve the hybridization performance of the probes. Hybridization results with specimens prepared by RNAsin and heat treatment were similar to those obtained after Nonidet P-40 and formaldehyde treatment.

A selection of microplates and an inverted microscope were used for the detection of immunofluorescence in large numbers of infected cell monolayers. The optical features of this instrument in combination with either Terasaki or Microlymp plates, simplify procedures and allow economical use of reagents, for determining fluorescent antibody levels. The techniques described were applied for estimating M and G immunoglobulins against rotavirus in elderly patients and the validity of the tests was established.

Summary The structural and intracellular proteins of the murine hepatitis viruses MHV-JHM and MHV-A59 were studied by tryptic peptide mapping. The results demonstrated that the virions contained three distinct proteins: the two related chains of the E2 complex, the nucleocapsid protein and the heterogeneous E1 complex. Five distinct virus-specific proteins were synthesized by infected cells. Three of the five intracellular proteins contained tryptic-peptides with properties similar to the three structural proteins. Models describing the evolution of the proteins are proposed. Although the pathogenic properties of MHV-JHM and MHV-A59 differ greatly, the tryptic peptide maps of the corresponding proteins of these MHV strains were remarkably similar.