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Sample records for queen cell virus

  1. New evidence that Deformed Wing Virus and Black Queen Cell Virus are Multi-host pathogens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The host-range breadth of pathogens can have important consequences for pathogens’ long term evolution and virulence, and play critical roles in the emergence and spread of the new diseases. Black queen cell virus (BQCV) and Deformed wing virus (DWV) are the two most common and prevalent viruses in...

  2. [Research Progress in Black Queen Cell Virus Causing Disease].

    PubMed

    Yang, Qian; Zhang, Jian; Song, Zhanyun; Zheng, Yan; Wang, Xianghui; Sui, Jiachen; Wang, Zhenguo; Mou, Jun

    2015-05-01

    In nature, honeybees are the most important pollinators. They play a vital role in both protecting the diversity of natural ecosystems, and maintaining the yield-improving effects of agroecosystems. But in recent years, epidemic disease in bees has caused huge losses. Black Queen Cell Virus (BQCV) is a bee pathogen that was first reported in 1955. It mainly infects bee larvae and pupae, making their bodies turn dark and black, and causing a massive decrease in the bee population. More specifically, the virus makes the exterior of the cell walls in the larvae and pupae turn black. BQCV is a seasonal epidemic, spread by means horizontal and vertical transmission, and is often unapparent. BQCV not only infects a variety of bee species, but also spiders, centipedes and other arthropods. It can also be coinfected with other honeybee viruses. In recent years, research has shown that the Nosema intestinal parasite plays an important role in BQCV transmission and bees carrying Nosema that become infected with BQCV have increased mortality. Here we summarize current research on the incidence, prevalence, geographical distribution and transmission of BQCV. PMID:26470541

  3. Host Range Expansion of Honey Bee Black Queen Cell Virus in the Bumble Bee, Bombus huntii

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Honey bee viruses display a host range that is not restricted to their original host, European honey bees, Apis mellifera. Here we provide the first evidence that Black Queen Cell Virus (BQCV), one of the most prevalent honey bee viruses, can cause an infection in both laboratory-reared and field-co...

  4. [Symptomatic Black Queen Cell Virus infection of drone brood in Hessian apiaries].

    PubMed

    Siede, Reinhold; Büchler, Ralph

    2003-01-01

    The Black Queen Cell Virus (BQCV) can affect brood of the honey bee (Apis mellifera). In general queen cells are endangered showing dark coloured cell walls as typical symptoms. Worker- and dronebrood can be infected by BQCV but normally without clinical symptoms. This paper describes for the first time a symptomatic BQCV-infection of diseased drone brood found on two bee yards in Hessen/Germany in 2001. The drone larvae were seriously damaged and some of them were dead. Samples of the affected brood were tested for BQCV by the PCR detection method. A BQCV specific nucleic acid fragment was found. The PCR product were sequenced and aligned with the relevant GenBank entry. At the nucleic acid level as well as at the deduced protein level the isolate showed a high similarity with the south african isolate noted in GenBank. PMID:12680279

  5. Detection of Acute Bee Paralysis Virus and Black Queen Cell Virus from Honeybees by Reverse Transcriptase PCR

    PubMed Central

    Benjeddou, Mongi; Leat, Neil; Allsopp, Mike; Davison, Sean

    2001-01-01

    A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3′ end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV. PMID:11319129

  6. Acceptance of mated Queens and Queen Cells in Colonies of Russian and Italian Honey Bees

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Requeening colonies is a standard beekeeping practice with both mated queens and queen cells. More beekeepers are requeening with Russian honey bees queens because of their significantly higher resistance to varroa and tracheal mites, their good honey production and their overwintering abilities. ...

  7. Deformed wing virus can be transmitted during natural mating in honey bees and infect the queens.

    PubMed

    Amiri, Esmaeil; Meixner, Marina D; Kryger, Per

    2016-01-01

    Deformed wing virus is an important contributor to honey bee colony losses. Frequently queen failure is reported as a cause for colony loss. Here we examine whether sexual transmission during multiple matings of queens is a possible way of virus infection in queens. In an environment with high prevalence of deformed wing virus, queens (n = 30) were trapped upon their return from natural mating flights. The last drone's endophallus (n = 29), if present, was removed from the mated queens for deformed wing virus quantification, leading to the detection of high-level infection in 3 endophalli. After oviposition, viral quantification revealed that seven of the 30 queens had high-level deformed wing virus infections, in all tissues, including the semen stored in the spermathecae. Two groups of either unmated queens (n = 8) with induced egg laying, or queens (n = 12) mated in isolation with drones showing comparatively low deformed wing virus infections served as control. None of the control queens exhibited high-level viral infections. Our results demonstrate that deformed wing virus infected drones are competitive to mate and able to transmit the virus along with semen, which occasionally leads to queen infections. Virus transmission to queens during mating may be common and can contribute noticeably to queen failure. PMID:27608961

  8. Deformed wing virus can be transmitted during natural mating in honey bees and infect the queens

    PubMed Central

    Amiri, Esmaeil; Meixner, Marina D.; Kryger, Per

    2016-01-01

    Deformed wing virus is an important contributor to honey bee colony losses. Frequently queen failure is reported as a cause for colony loss. Here we examine whether sexual transmission during multiple matings of queens is a possible way of virus infection in queens. In an environment with high prevalence of deformed wing virus, queens (n = 30) were trapped upon their return from natural mating flights. The last drone’s endophallus (n = 29), if present, was removed from the mated queens for deformed wing virus quantification, leading to the detection of high-level infection in 3 endophalli. After oviposition, viral quantification revealed that seven of the 30 queens had high-level deformed wing virus infections, in all tissues, including the semen stored in the spermathecae. Two groups of either unmated queens (n = 8) with induced egg laying, or queens (n = 12) mated in isolation with drones showing comparatively low deformed wing virus infections served as control. None of the control queens exhibited high-level viral infections. Our results demonstrate that deformed wing virus infected drones are competitive to mate and able to transmit the virus along with semen, which occasionally leads to queen infections. Virus transmission to queens during mating may be common and can contribute noticeably to queen failure. PMID:27608961

  9. The effects of pesticides on queen rearing and virus titers in honey bees (Apis mellifera L.)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of sublethal pesticide exposure on queen emergence and virus titers were examined. Queen rearing colonies were fed pollen with chlorpyrifos (CPF) alone (pollen-1) and with CPF and the fungicide Pristine® (pollen-2). Fewer queens emerged when larvae from open foraging (i.e., outside) colo...

  10. Chronic Bee Paralysis Virus in Honeybee Queens: Evaluating Susceptibility and Infection Routes

    PubMed Central

    Amiri, Esmaeil; Meixner, Marina; Büchler, Ralph; Kryger, Per

    2014-01-01

    Chronic bee paralysis virus (CBPV) is known as a disease of worker honey bees. To investigate pathogenesis of the CBPV on the queen, the sole reproductive individual in a colony, we conducted experiments regarding the susceptibility of queens to CBPV. Results from susceptibility experiment showed a similar disease progress in the queens compared to worker bees after infection. Infected queens exhibit symptoms by Day 6 post infection and virus levels reach 1011 copies per head. In a transmission experiment we showed that social interactions may affect the disease progression. Queens with forced contact to symptomatic worker bees acquired an overt infection with up to 1011 virus copies per head in six days. In contrast, queens in contact with symptomatic worker bees, but with a chance to receive food from healthy bees outside the cage appeared healthy. The virus loads did not exceed 107 in the majority of these queens after nine days. Symptomatic worker bees may transmit sufficient active CBPV particles to the queen through trophallaxis, to cause an overt infection. PMID:24618857

  11. Queen pheromone regulates programmed cell death in the honey bee worker ovary.

    PubMed

    Ronai, I; Oldroyd, B P; Vergoz, V

    2016-10-01

    In social insect colonies the presence of a queen, secreting her pheromones, is a key environmental cue for regulating the reproductive state of workers. However, until recently the proximate molecular mechanisms underlying facultative worker sterility were unidentified. Studies into worker oogenesis in the honey bee (Apis mellifera) have indicated that programmed cell death is central to the regulation of oogenesis. Here we investigate how queen pheromone, age of the worker and ovary state affect both programmed cell death and cell number in worker ovaries. We describe a novel method to simultaneously measure programmed cell death (caspase activity) and live cell number (estimated from the amount of adenosine triphosphate) in an insect tissue. Workers exposed to queen pheromone have higher levels of caspase activity in the ovary than those not exposed. Our results suggest that queen pheromone triggers programmed cell death at the mid-oogenesis checkpoint causing the abortion of worker oocytes and reproductive inhibition of the worker caste. Nonetheless, high caspase activity is present in activated ovaries from workers not exposed to queen pheromone. This caspase activity is most likely to be from the nurse cells undergoing programmed cell death, in late oogenesis, for normal oocyte development. Our study shows that the social environment of an organism can influence programmed cell death within a tissue. PMID:27321063

  12. Dynamic changes in host-virus interactions associated with colony founding and social environment in fire ant queens (Solenopsis invicta).

    PubMed

    Manfredini, Fabio; Shoemaker, DeWayne; Grozinger, Christina M

    2016-01-01

    The dynamics of host-parasite interactions can change dramatically over the course of a chronic infection as the internal (physiological) and external (environmental) conditions of the host change. When queens of social insects found a colony, they experience changes in both their physiological state (they develop their ovaries and begin laying eggs) and the social environment (they suddenly stop interacting with the other members of the mother colony), making this an excellent model system for examining how these factors interact with chronic infections. We investigated the dynamics of host-viral interactions in queens of Solenopsis invicta (fire ant) as they transition from mating to colony founding/brood rearing to the emergence of the first workers. We examined these dynamics in naturally infected queens in two different social environments, where queens either founded colonies as individuals or as pairs. We hypothesized that stress associated with colony founding plays an important role in the dynamics of host-parasite interactions. We also hypothesized that different viruses have different modalities of interaction with the host that can be quantified by physiological measures and genomic analysis of gene expression in the host. We found that the two most prevalent viruses, SINV-1 and SINV-2, are associated with different fitness costs that are mirrored by different patterns of gene expression in the host. In fact SINV-2, the virus that imposes the significant reduction of a queen's reproductive output is also associated with larger changes of global gene expression in the host. These results show the complexity of interactions between S. invicta and two viral parasites. Our findings also show that chronic infections by viral parasites in insects are dynamic processes that may pose different challenges in the host, laying the groundwork for interesting ecological and evolutionary considerations. PMID:26811788

  13. Drama queens.

    PubMed

    Taylor, J

    1998-01-01

    Abstract Why is it that many 'lesbian playwrights' are unwilling to define themselves as such? "Drama Queens: Ruling with a Rod of Irony" attempts to answer this question and to discover what the term 'lesbian playwright' means within contemporary culture. It dissects the dominant homophobic and misogynist mythologies that have outlawed 'queer' girl writers to the underskirts of British theatre, and ultimately denied them either artistic or commercial currency. It examines the history of the label in the context of feminism, gay liberation and positive representation, and queries its aesthetic and economic viability in a climate where the 'lesbian playwright' is not even supported by her own community. Finally, it is an exploration into radical forms, working methodologies and new genres stimulated by being neither semantic Man nor Woman. It is a piece about cultural terrorism-and how to avoid capture. PMID:24785519

  14. Cell injury with viruses.

    PubMed Central

    Tamm, I.

    1975-01-01

    The inhibitions of cellular protein and RNA synthesis in picornavirus-infected cells are early events which require only limited synthesis of virus-specific proteins. The inhibition of cellular protein synthesis appears to be due to blocking of the association of ribosomes with host messenger RNA. Inhibition of cellular RNA synthesis involves inactivation of the template-enzyme complex and affects ribosomal RNA synthesis before messenger RNA synthesis. Inhibition of cellular DNA synthesis also occurs early and may be secondary to inhibition of cellular protein synthesis. Early chromatid breaks may be related to inhibitions of cellular protein and nucleic acid synthesis. Stimulation of phospholipid synthesis in picornavirus-infected cells also requires only limited synthesis of virus-specific proteins. In contrast, release of lysosomal enzymes, proliferation of smooth cytoplasmic membranes, cellular vacuolization, retraction, and rounding, and diffuse chromosomal changes related to karyorrhexis and pyknosis are all dependent on considerable synthesis of virus-specific proteins during the middle part of the picornavirus growth cycle. The early virus-induced alterations in cellular biosynthetic processes are not the direct or sole cause of the subsequent marked pathologic changes in the membranes and chromosomes of the cell. Images Figure 1 PMID:1180330

  15. The Eight Queens Problem.

    ERIC Educational Resources Information Center

    Olson, Alton T.

    1993-01-01

    Presents a series of solution methods to the Eight Queens Problem of placing eight queens on a chess board so that no one queen can capture another. Solution methods progress from empirical approaches to the use of computer algorithms. Geometric transformations are used to find other solutions. (MDH)

  16. Virus Cell-to-Cell Transmission▿

    PubMed Central

    Mothes, Walther; Sherer, Nathan M.; Jin, Jing; Zhong, Peng

    2010-01-01

    Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion molecules, and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections. PMID:20375157

  17. Factors influencing survival duration and choice of virgin queens in the stingless bee Melipona quadrifasciata

    NASA Astrophysics Data System (ADS)

    Kärcher, Martin H.; Menezes, Cristiano; Alves, Denise A.; Beveridge, Oliver S.; Imperatriz-Fonseca, Vera-Lucia; Ratnieks, Francis L. W.

    2013-06-01

    In Melipona quadrifasciata, about 10 % of the females develop into queens, almost all of which are killed. Occasionally, a new queen replaces or supersedes the mother queen or heads a new colony. We investigated virgin queen fate in queenright and queenless colonies to determine the effects of queen behaviour, body mass, nestmate or non-nestmate status, queenright or queenless colony status, and, when queenless, the effect of the time a colony had been queenless, on survival duration and acceptance. None of 220 virgin queens observed in four observation hives ever attacked another virgin queen nor did any of 88 virgin queens introduced into queenright colonies ever attack the resident queen. A new queen was only accepted in a queenless colony. Factors increasing survival duration and acceptance of virgin queens were to emerge from its cell at 2 h of queenlessness, to hide, and to avoid fights with workers. In this way, a virgin queen was more likely to be available when a colony chooses a new queen, 24-48 h after resident queen removal. Running, walking or resting, antennating or trophallaxis, played little or no role, as did the factors body mass or nestmate. "Queen choice" took about 2 h during which time other virgin queens were still being killed by workers. During this agitated process, the bees congregated around the new queen. She inflated her abdomen and some of the workers deposited a substance on internal nest surfaces including the glass lid of the observation hive.

  18. Patterns of viral infection in honey bee queens.

    PubMed

    Francis, Roy Mathew; Nielsen, Steen Lykke; Kryger, Per

    2013-03-01

    The well-being of a colony and replenishment of the workers depends on a healthy queen. Diseases in queens are seldom reported, and our knowledge on viral infection in queens is limited. In this study, 86 honey bee queens were collected from beekeepers in Denmark. All queens were tested separately by two real-time PCRs: one for the presence of deformed wing virus (DWV), and one that would detect sequences of acute bee-paralysis virus, Kashmir bee virus and Israeli acute paralysis virus (AKI complex). Worker bees accompanying the queen were also analysed. The queens could be divided into three groups based on the level of infection in their head, thorax, ovary, intestines and spermatheca. Four queens exhibited egg-laying deficiency, but visually all queens appeared healthy. Viral infection was generally at a low level in terms of AKI copy numbers, with 134/430 tissues (31 %) showing the presence of viral infection ranging from 10(1) to 10(5) copies. For DWV, 361/340 tissues (84 %) showed presence of viral infection (DWV copies ranging from 10(2) to 10(12)), with 50 tissues showing viral titres >10(7) copies. For both AKI and DWV, the thorax was the most frequently infected tissue and the ovaries were the least frequently infected. Relative to total mass, the spermatheca showed significantly higher DWV titres than the other tissues. The ovaries had the lowest titre of DWV. No significant differences were found among tissues for AKI. A subsample of 14 queens yielded positive results for the presence of negative-sense RNA strands, thus demonstrating active virus replication in all tissues. PMID:23223622

  19. Patterns of viral infection in honey bee queens

    PubMed Central

    Francis, Roy Mathew; Nielsen, Steen Lykke

    2013-01-01

    The well-being of a colony and replenishment of the workers depends on a healthy queen. Diseases in queens are seldom reported, and our knowledge on viral infection in queens is limited. In this study, 86 honey bee queens were collected from beekeepers in Denmark. All queens were tested separately by two real-time PCRs: one for the presence of deformed wing virus (DWV), and one that would detect sequences of acute bee-paralysis virus, Kashmir bee virus and Israeli acute paralysis virus (AKI complex). Worker bees accompanying the queen were also analysed. The queens could be divided into three groups based on the level of infection in their head, thorax, ovary, intestines and spermatheca. Four queens exhibited egg-laying deficiency, but visually all queens appeared healthy. Viral infection was generally at a low level in terms of AKI copy numbers, with 134/430 tissues (31 %) showing the presence of viral infection ranging from 101 to 105 copies. For DWV, 361/340 tissues (84 %) showed presence of viral infection (DWV copies ranging from 102 to 1012), with 50 tissues showing viral titres >107 copies. For both AKI and DWV, the thorax was the most frequently infected tissue and the ovaries were the least frequently infected. Relative to total mass, the spermatheca showed significantly higher DWV titres than the other tissues. The ovaries had the lowest titre of DWV. No significant differences were found among tissues for AKI. A subsample of 14 queens yielded positive results for the presence of negative-sense RNA strands, thus demonstrating active virus replication in all tissues. PMID:23223622

  20. 14. Hell Gate Bridge south abutment tower. Queens, Queens Co., ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    14. Hell Gate Bridge south abutment tower. Queens, Queens Co., NY. Sec. 4207, MP 7.29. - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  1. Queen signals in a stingless bee: suppression of worker ovary activation and spatial distribution of active compounds

    PubMed Central

    Nunes, Túlio M.; Mateus, Sidnei; Favaris, Arodi P.; Amaral, Mônica F. Z. J.; von Zuben, Lucas G.; Clososki, Giuliano C.; Bento, José M. S.; Oldroyd, Benjamin P.; Silva, Ricardo; Zucchi, Ronaldo; Silva, Denise B.; Lopes, Norberto P.

    2014-01-01

    In most species of social insect the queen signals her presence to her workers via pheromones. Worker responses to queen pheromones include retinue formation around the queen, inhibition of queen cell production and suppression of worker ovary activation. Here we show that the queen signal of the Brazilian stingless bee Friesella schrottkyi is a mixture of cuticular hydrocarbons. Stingless bees are therefore similar to ants, wasps and bumble bees, but differ from honey bees in which the queen's signal mostly comprises volatile compounds originating from the mandibular glands. This shows that cuticular hydrocarbons have independently evolved as the queen's signal across multiple taxa, and that the honey bees are exceptional. We also report the distribution of four active queen-signal compounds by Matrix-assisted laser desorption/ionization (MALDI) imaging. The results indicate a relationship between the behavior of workers towards the queen and the likely site of secretion of the queen's pheromones. PMID:25502598

  2. 'Snow Queen' Animation

    NASA Technical Reports Server (NTRS)

    2008-01-01

    This animation consists of two close-up images of 'Snow Queen,' taken several days apart, by the Robotic Arm Camera (RAC) aboard NASA's Phoenix Mars Lander.

    Snow Queen is the informal name for a patch of bright-toned material underneath the lander.

    Thruster exhaust blew away surface soil covering Snow Queen when Phoenix landed on May 25, 2008, exposing this hard layer comprising several smooth rounded cavities beneath the lander. The RAC images show how Snow Queen visibly changed between June 15, 2008, the 21st Martian day, or sol, of the mission and July 9, 2008, the 44th sol.

    Cracks as long as 10 centimeters (about four inches) appeared. One such crack is visible at the left third and the upper third of the Sol 44 image. A seven millimeter (one-third inch) pebble or clod appears just above and slightly to the right of the crack in the Sol 44 image. Cracks also appear in the lower part of the left third of the image. Other pieces noticeably shift, and some smooth texture has subtly roughened.

    The Phoenix team carefully positioned and focused RAC the same way in both images. Each image is about 60 centimeters, or about two feet, wide. The object protruding in from the top on the right half of the images is Phoenix's thermal and electrical conductivity probe.

    Snow Queen and other ice exposed by Phoenix landing and trenching operations on northern polar Mars is the first time scientists have been able to monitor Martian ice at a place where temperatures are cold enough that the ice doesn't immediately sublimate, or vaporize, away.

    The Phoenix Mission is led by the University of Arizona, Tucson, on behalf of NASA. Project management of the mission is by NASA's Jet Propulsion Laboratory, Pasadena, Calif. Spacecraft development is by Lockheed Martin Space Systems, Denver.

  3. DNA Tumor Viruses and Cell Metabolism

    PubMed Central

    Mushtaq, Muhammad; Darekar, Suhas

    2016-01-01

    Viruses play an important role in cancerogenesis. It is estimated that approximately 20% of all cancers are linked to infectious agents. The viral genes modulate the physiological machinery of infected cells that lead to cell transformation and development of cancer. One of the important adoptive responses by the cancer cells is their metabolic change to cope up with continuous requirement of cell survival and proliferation. In this review we will focus on how DNA viruses alter the glucose metabolism of transformed cells. Tumor DNA viruses enhance “aerobic” glycolysis upon virus-induced cell transformation, supporting rapid cell proliferation and showing the Warburg effect. Moreover, viral proteins enhance glucose uptake and controls tumor microenvironment, promoting metastasizing of the tumor cells. PMID:27034740

  4. [The oral problems of queen Elizabeth I].

    PubMed

    Eijkman, M A J

    2012-05-01

    Queen Elizabeth I of England (1533-1603), probably the most famous English Queen ever, had persistent oral problems. Her oral problems were so serious that they probably hampered the Queen in the performance of her tasks. PMID:22667195

  5. Interaction and behavior of virgin and physogastric queens in three Meliponini species (Hymenoptera, Apidae).

    PubMed

    Nogueira-Ferreira, F H; Silva-Matos, E V; Zucchi, R

    2009-01-01

    We studied the behavior of virgin queens of the stingless bee species Schwarziana quadripunctata, Paratrigona lineata and Tetragona clavipes, investigating internal nest activities, including the cell provisioning and oviposition process. We made direct observation of queen behavior, with the aid of video filming. Forty-four virgin queens of S. quadripunctata were observed; one was larger and more attractive than the others. Miniature queens were more abundant than normal-size queens; both were found in prison chambers. Agonistic behavior between virgin and physogastric queens of P. lineata was observed during attempts at queen supersedure. After the disappearance of the physogastric queen and the appearance of a virgin queen in T. clavipes nests, the brood cells were sealed with pollen alone, but no egg. In all three species, the presence of one or more virgin queens appeared to make the colonies nervous, even though constant production of virgin queens is vital to the survival of the colony and is part of the colony cycle in these bees. PMID:19554769

  6. 13. New York Connecting RR: Hell Gate Bridge. Queens, Queens ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    13. New York Connecting RR: Hell Gate Bridge. Queens, Queens Co., NY. Sec. 4207, MP 7.29. (See HAER No. NY-88 for further documentation on this site). - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  7. 11. New York Connecting RR: Hell Gate Bridge. Queens, Queens ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    11. New York Connecting RR: Hell Gate Bridge. Queens, Queens Co., NY. Sec. 4207, MP 7.29. (See HAER No. NY-88 for further documentation on this site). - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  8. 12. New York Connecting RR: Hell Gate Bridge. Queens, Queens ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    12. New York Connecting RR: Hell Gate Bridge. Queens, Queens Co., NY. Sec. 4207, MP 7.29. (See HAER No. NY-88 for further documentation on this site). - Northeast Railroad Corridor, Amtrak Route between New Jersey/New York & New York/Connecticut State Lines, New York County, NY

  9. Targeting cancer stem cells with oncolytic virus

    PubMed Central

    Tong, Yin

    2014-01-01

    Cancer stem cells (CSCs) represent a distinct subpopulation of cancer cells which are shown to be relatively resistant to conventional anticancer therapies and have been correlated to disease recurrence. Oncolytic viruses utilize methods of cell killing that differ from traditional therapies and thus are able to elude the typical mechanisms that CSCs use to resist current chemotherapies and radiotherapies. Moreover, genetically engineered oncolytic viruses may further augment the oncolytic effects. Here we review the recent data regarding the ability of several oncolytic viruses to eradicate CSCs.

  10. Neural stem cells attacked by Zika virus.

    PubMed

    Nguyen, Ha Nam; Qian, Xuyu; Song, Hongjun; Ming, Guo-Li

    2016-07-01

    The current outbreak of Zika virus-associated diseases in South America and its threat to spread to other parts of the world has emerged as a global health emergency. Insights from cell and animal models to understand how Zika virus causes severe birth defects may lead to treatments and prevention of these diseases. PMID:27283801

  11. Cell Culture for Production of Insecticidal Viruses.

    PubMed

    Reid, Steven; Chan, Leslie C L; Matindoost, Leila; Pushparajan, Charlotte; Visnovsky, Gabriel

    2016-01-01

    While large-scale culture of insect cells will need to be conducted using bioreactors up to 10,000 l scale, many of the main challenges for cell culture-based production of insecticidal viruses can be studied using small-scale (20-500 ml) shaker/spinner flasks, either in free suspension or using microcarrier-based systems. These challenges still relate to the development of appropriate cell lines, stability of virus strains in culture, enhancing virus yields per cell, and the development of serum-free media and feeds for the desired production systems. Hence this chapter presents mainly the methods required to work with and analyze effectively insect cell systems using small-scale cultures. Outlined are procedures for quantifying cells and virus and for establishing frozen cells and virus stocks. The approach for maintaining cell cultures and the multiplicity of infection (MOI) and time of infection (TOI) parameters that should be considered for conducting infections are discussed.The methods described relate, in particular, to the suspension culture of Helicoverpa zea and Spodoptera frugiperda cell lines to produce the baculoviruses Helicoverpa armigera nucleopolyhedrovirus, HearNPV, and Anticarsia gemmatalis multicapsid nucleopolyhedrovirus, AgMNPV, respectively, and the production of the nonoccluded Oryctes nudivirus, OrNV, using an adherent coleopteran cell line. PMID:27565495

  12. Ozone inactivation of cell-associated viruses.

    PubMed Central

    Emerson, M A; Sproul, O J; Buck, C E

    1982-01-01

    The inactivation of HEp-2 cell-associated poliovirus (Sabin 1) and coxsackievirus A9 was investigated in three experimental systems, using ozone as a disinfectant. The cell-associated viral samples were adjusted to a turbidity of 5 nephelometric turbidity units. The cell-associated poliovirus and coxsackievirus samples demonstrated survival in a continuous-flow ozonation system at applied ozone dosages of 4.06 and 4.68 mg/liter, respectively, for 30 s. Unassociated viral controls were inactivated by the application of 0.081 mg of ozone per liter for 10 s. Ultrasonic treatment of cell-associated enteric viruses did not increase inactivation of the cell-associated viruses. The batch reactor with a declining ozone residual did not effect total inactivation of either cell-associated enteric virus. These cell-associated viruses were completely inactivated after exposure to ozone in a batch reactor using continuous ozonation. Inactivation of cell-associated poliovirus required a 2-min contact period with an applied ozone dosage of 6.82 mg/liter and a residual ozone concentration of 4.70 mg/liter, whereas the coxsackievirus was completely inactivated after a 5-min exposure to an applied ozone dosage of 4.81 mg/liter with an ozone residual of 2.18 mg/liter. These data indicate that viruses associated with cells or cell fragments are protected from inactivation by ozone concentrations that readily inactivate purified virus. The cell-associated viral samples used in this research contained particles that were 10 to 15 microns in size. Use of a filtration system before ozonation would remove these particles, thereby facilitating inactivation of any remaining viruses associated with cellular fragments. PMID:6280611

  13. Rescue of rous sarcoma virus from rous sarcoma virus-transformed mammalian cells.

    PubMed

    Coffin, J M

    1972-07-01

    Rat cells transformed by the B77 strain of avian sarcoma virus produce no virus-like particles, yet B77 virus was rescued from these cells by Sendai virus-mediated fusion with chicken cells. This virus rescue was not affected by treatment of the chicken cells with agents that rendered the cells incapable of dividing, although such treatment greatly reduced the ability of the chicken cells to plate as infectious centers after infection with B77 virus. Fusion of R(B77) cells with chicken erythrocytes also led to virus rescue, although with less efficiency than fusion with chicken fibroblasts. Therefore, virus rescue was probably due to a factor or factors contributed by chicken cells which aid in virus production. PMID:4339192

  14. Cell entry of hepatitis C virus

    SciTech Connect

    Bartosch, Birke . E-mail: Birke.Bartosch@ens-lyon.fr; Cosset, Francois-Loic . E-mail: Francois-Loic.Cosset@ens-lyon.fr

    2006-04-25

    Hepatitis C virus (HCV), an important human pathogen, is an enveloped, positive-stranded RNA virus classified in the hepacivirus genus of the Flaviviridae family. Cell attachment of flaviviruses generally leads to endocytosis of bound virions. Systems that support HCV replication and particle formation in vitro are emerging only now, 16 years after the discovery of the virus. Albeit this limitation, the route of HCV cell entry as well as 'capture' molecules involved in low-affinity interactions for the initial contact of HCV with target cells and potential high-affinity receptor candidates that may mediate HCV trafficking and fusion has been described. The objective of this review is to summarize the contribution of different HCV model systems to our current knowledge about structure of the HCV GPs E1 and E2 and their roles in cell entry comprising cell attachment, interactions with cellular receptors, endocytosis, and fusion.

  15. Psoralen inactivation of influenza and herpes simplex viruses and of virus-infected cells

    SciTech Connect

    Redfield, D.C.; Richman, D.D.; Oxman, M.N.; Kronenberg, L.H.

    1981-06-01

    Psoralen compounds covalently bind to nucleic acids when irradiated with long-wavelength ultraviolet light. This treatment can destroy the infectivity of deoxyribonucleic acid and ribonucleic acid viruses. Two psoralen compounds, 4'-hydroxymethyltrioxsalen and 4'-aminomethyltrioxsalen, were used with long-wavelength ultraviolet light to inactivate cell-free herpes simplex and influenza viruses and to render virus-infected cells noninfectious. This method of inactivation was compared with germicidal (short-wavelength) ultraviolet light irradiation. The antigenicity of the treated, virus-infected, antigen-bearing cells was examined by immunofluorescence and radioimmunoassay and by measuring the capacity of the herpes simplex virus-infected cells to stimulate virus-specific lymphocyte proliferation. The infectivity of the virus-infected cells could be totally eliminated without altering their viral antigenicity. The use of psoralen plus long-wavelength ultraviolet light is well suited to the preparation of noninfectious virus antigens and virus antigen-bearing cells for immunological assays.

  16. Cells in Dengue Virus Infection In Vivo

    PubMed Central

    Noisakran, Sansanee; Onlamoon, Nattawat; Songprakhon, Pucharee; Hsiao, Hui-Mien; Chokephaibulkit, Kulkanya; Perng, Guey Chuen

    2010-01-01

    Dengue has been recognized as one of the most important vector-borne emerging infectious diseases globally. Though dengue normally causes a self-limiting infection, some patients may develop a life-threatening illness, dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS). The reason why DHF/DSS occurs in certain individuals is unclear. Studies in the endemic regions suggest that the preexisting antibodies are a risk factor for DHF/DSS. Viremia and thrombocytopenia are the key clinical features of dengue virus infection in patients. The amounts of virus circulating in patients are highly correlated with severe dengue disease, DHF/DSS. Also, the disturbance, mainly a transient depression, of hematological cells is a critical clinical finding in acute dengue patients. However, the cells responsible for the dengue viremia are unresolved in spite of the intensive efforts been made. Dengue virus appears to replicate and proliferate in many adapted cell lines, but these in vitro properties are extremely difficult to be reproduced in primary cells or in vivo. This paper summarizes reports on the permissive cells in vitro and in vivo and suggests a hematological cell lineage for dengue virus infection in vivo, with the hope that a new focus will shed light on further understanding of the complexities of dengue disease. PMID:22331984

  17. Killing and replacing queen-laid eggs: low cost of worker policing in the honeybee.

    PubMed

    Kärcher, Martin H; Ratnieks, Francis L W

    2014-07-01

    Worker honeybees, Apis mellifera, police each other's reproduction by killing worker-laid eggs. Previous experiments demonstrated that worker policing is effective, killing most (∼98%) worker-laid eggs. However, many queen-laid eggs were also killed (∼50%) suggesting that effective policing may have high costs. In these previous experiments, eggs were transferred using forceps into test cells, mostly into unrelated discriminator colonies. We measured both the survival of unmanipulated queen-laid eggs and the proportion of removal errors that were rectified by the queen laying a new egg. Across 2 days of the 3-day egg stage, only 9.6% of the queen-laid eggs in drone cells and 4.1% in worker cells were removed in error. When queen-laid eggs were removed from cells, 85% from drone cells and 61% from worker cells were replaced within 3 days. Worker policing in the honeybee has a high benefit to policing workers because workers are more related to the queen's sons (brothers, r = 0.25) than sister workers' sons (0.15). This study shows that worker policing also has a low cost in terms of the killing of queen-laid eggs, as only a small proportion of queen-laid eggs are killed, most of which are rapidly replaced. PMID:24921604

  18. Queen reproductive state modulates pheromone production and queen-worker interactions in honeybees

    PubMed Central

    Kocher, Sarah D.; Richard, Freddie-Jeanne; Tarpy, David R.

    2009-01-01

    The mandibular glands of queen honeybees produce a pheromone that modulates many aspects of worker honeybee physiology and behavior and is critical for colony social organization. The exact chemical blend produced by the queen differs between virgin and mated, laying queens. Here, we investigate the role of mating and reproductive state on queen pheromone production and worker responses. Virgin queens, naturally mated queens, and queens instrumentally inseminated with either semen or saline were collected 2 days after mating or insemination. Naturally mated queens had the most activated ovaries and the most distinct chemical profile in their mandibular glands. Instrumentally inseminated queens were intermediate between virgins and naturally mated queens for both ovary activation and chemical profiles. There were no significant differences between semen- and saline-inseminated queens. Workers were preferentially attracted to the mandibular gland extracts from queens with significantly more activated ovaries. These studies suggest that the queen pheromone blend is modulated by the reproductive status of the queens, and workers can detect these subtle differences and are more responsive to queens with higher reproductive potential. Furthermore, it appears as if insemination substance does not strongly affect physiological characteristics of honeybee queens 2 days after insemination, suggesting that the insemination process or volume is responsible for stimulating these early postmating changes in honeybee queens. PMID:22476212

  19. Cell carriers for oncolytic viruses: current challenges and future directions.

    PubMed

    Roy, Dominic G; Bell, John C

    2013-01-01

    The optimal route for clinical delivery of oncolytic viruses is thought to be systemic intravenous injection; however, the immune system is armed with several highly efficient mechanisms to remove pathogens from the circulatory system. To overcome the challenges faced in trying to delivery oncolytic viruses specifically to tumors via the bloodstream, carrier cells have been investigated to determine their suitability as delivery vehicles for systemic administration of oncolytic viruses. Cell carriers protect viruses from neutralization, one of the most limiting aspects of oncolytic virus interaction with the immune system. Cell carriers can also possess inherent tumor tropism, thus directing the delivery of the virus more specifically to a tumor. With preclinical studies already demonstrating the success and feasibility of this approach with multiple oncolytic viruses, clinical evaluation of cell-mediated delivery of viruses is on the horizon. Meanwhile, ongoing preclinical studies are aimed at identifying new cellular vehicles for oncolytic viruses and improving current promising cell carrier platforms. PMID:27512657

  20. Monitoring virus entry into living cells using DiD-labeled dengue virus particles.

    PubMed

    Ayala-Nuñez, Nilda V; Wilschut, Jan; Smit, Jolanda M

    2011-10-01

    A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular trafficking behavior, and the moment of membrane fusion of single virus particles in living cells. Here we describe the development and applications of a single-virus tracking assay based on the use of DiD-labeled dengue virus (DENV) in BS-C-1 cells. In addition - and using the same experimental setup - we present a binding and fusion assay that can be used to obtain a rapid insight into the relative extent of virus binding to the cell surface and membrane fusion. Details of virus labeling and characterization, microscopy setup, protocols, data analysis, and hints for troubleshooting are described throughout the paper. PMID:21855634

  1. Transformation of rat liver cells with chicken sarcoma virus B77 and murine sarcoma virus.

    PubMed

    Altaner, C; Hlavayova, E

    1973-02-01

    Rat liver cells in vitro were transformed with chicken sarcoma virus B77, giving RL(B77) cells, and with murine sarcoma virus (Harvey), giving RL(MSV) cells. Rat liver cells transformed spontaneously in vitro were designated RL cells. In addition, the RL(MSV) cell line was adapted for growth in culture fluid containing 25 mug of 5-bromodeoxyuridine per ml. All cell lines were tumorigenic in 1-wk-old rats. The number of cells needed for induction of tumor growth was 1,000-fold higher in the case of RL(B77) cells in comparison with RL(MSV) cells and RL cells. No production of viral particles from any of the cell lines investigated was detected by plating concentrated supernatant fluid of the cultures on different secondary embryo cells with and without fusion by Sendai virus, by labeling with uridine-5-(3)H, or by assay for deoxyribonucleic acid polymerase activity. The viral genome was rescued by fusion of RL(B77) cells with chicken cells. Chicken sarcoma virus rescued from (RL(B77) cells differed in plating efficiency on duck cells from B77 virus rescued from transformed rat embryo cells. No virus was rescued after fusion of RL(MSV) and RL cells with mouse, rat, or chicken embryo cells. Infectious murine sarcoma virus can be induced by 5-bromodeoxyuridine from RL(MSV) cells. PMID:4347422

  2. Measles Virus Induces Functional TRAIL Production by Human Dendritic Cells

    PubMed Central

    Vidalain, Pierre-Olivier; Azocar, Olga; Lamouille, Barbara; Astier, Anne; Rabourdin-Combe, Chantal; Servet-Delprat, Christine

    2000-01-01

    Measles virus infection induces a profound immunosuppression that can lead to serious secondary infections. Here we demonstrate that measles virus induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA and protein expression in human monocyte-derived dendritic cells. Moreover, measles virus-infected dendritic cells are shown to be cytotoxic via the TRAIL pathway. PMID:10590149

  3. 'Queen of Hearts' Oakleaf Hydrangea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A late-blooming oakleaf hydrangea (Hydrangea quercifolia) cultivar was released by the U.S. National Arboretum. ‘Queen of Hearts’ has grown 6.5 feet high and 11 feet wide in 11 years. In early summer, it is covered with 11-inch-long inflorescences that are held upright above the foliage. Flowers ...

  4. CD4-Negative Cells Bind Human Immunodeficiency Virus Type 1 and Efficiently Transfer Virus to T Cells

    PubMed Central

    Olinger, Gene G.; Saifuddin, Mohammed; Spear, Gregory T.

    2000-01-01

    The ability of human immunodeficiency virus strain MN (HIVMN), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIVMN bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4+) and CD4-negative (CD4−) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4− cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4− cells was not due to stimulatory signals provided by CD4− cells or infection of CD4− cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4− cells is efficiently passed to CD4+ T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4− cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4− cells is an important route for infection of T cells in vivo. PMID:10954556

  5. Traditional and Modern Cell Culture in Virus Diagnosis.

    PubMed

    Hematian, Ali; Sadeghifard, Nourkhoda; Mohebi, Reza; Taherikalani, Morovat; Nasrolahi, Abbas; Amraei, Mansour; Ghafourian, Sobhan

    2016-04-01

    Cell cultures are developed from tissue samples and then disaggregated by mechanical, chemical, and enzymatic methods to extract cells suitable for isolation of viruses. With the recent advances in technology, cell culture is considered a gold standard for virus isolation. This paper reviews the evolution of cell culture methods and demonstrates why cell culture is a preferred method for identification of viruses. In addition, the advantages and disadvantages of both traditional and modern cell culture methods for diagnosis of each type of virus are discussed. Detection of viruses by the novel cell culture methods is considered more accurate and sensitive. However, there is a need to include some more accurate methods such as molecular methods in cell culture for precise identification of viruses. PMID:27169004

  6. Neonicotinoid pesticides severely affect honey bee queens

    PubMed Central

    Williams, Geoffrey R.; Troxler, Aline; Retschnig, Gina; Roth, Kaspar; Yañez, Orlando; Shutler, Dave; Neumann, Peter; Gauthier, Laurent

    2015-01-01

    Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances. PMID:26459072

  7. Neonicotinoid pesticides severely affect honey bee queens.

    PubMed

    Williams, Geoffrey R; Troxler, Aline; Retschnig, Gina; Roth, Kaspar; Yañez, Orlando; Shutler, Dave; Neumann, Peter; Gauthier, Laurent

    2015-01-01

    Queen health is crucial to colony survival of social bees. Recently, queen failure has been proposed to be a major driver of managed honey bee colony losses, yet few data exist concerning effects of environmental stressors on queens. Here we demonstrate for the first time that exposure to field-realistic concentrations of neonicotinoid pesticides during development can severely affect queens of western honey bees (Apis mellifera). In pesticide-exposed queens, reproductive anatomy (ovaries) and physiology (spermathecal-stored sperm quality and quantity), rather than flight behaviour, were compromised and likely corresponded to reduced queen success (alive and producing worker offspring). This study highlights the detriments of neonicotinoids to queens of environmentally and economically important social bees, and further strengthens the need for stringent risk assessments to safeguard biodiversity and ecosystem services that are vulnerable to these substances. PMID:26459072

  8. Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses.

    PubMed

    Roth, Bernhard; Mohr, Hannah; Enders, Martin; Garten, Wolfgang; Gregersen, Jens-Peter

    2012-01-11

    This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼10(4)), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses. PMID:22119922

  9. Transgenic cell lines for detection of animal viruses.

    PubMed Central

    Olivo, P D

    1996-01-01

    Rapid diagnostic assays based on direct detection of viral antigen or nucleic acid are being used with increasing frequency in clinical virology laboratories. Virus culture, however, remains the only way to detect infectious virus and to analyze clinically relevant viral phenotypes, such as drug resistance. Growth of viruses in cell culture is labor intensive and time-consuming and requires the use of many different cell lines. Transgenic technology, together with increasing knowledge of the molecular pathways of virus replication, offers the possibility of using genetically modified cell lines to improve virus growth in cell culture and to facilitate detection of virus-infected cells. Genetically modifying cells so that they express a reporter gene only after infection with a specific virus can allow the detection of infectious virus by rapid and simple enzyme assays such as beta-galactosidase assays without the need for antibodies. Although transgenic cells have recently been successfully used for herpes simplex virus detection, much more work needs to be done to adapt this technology to other human viral pathogens such as cytomegalovirus and respiratory viruses. This review offers some strategies for applying this technology to a wide spectrum of animal viruses. PMID:8809463

  10. Archaeal viruses at the cell envelope: entry and egress

    PubMed Central

    Quemin, Emmanuelle R. J.; Quax, Tessa E. F.

    2015-01-01

    The cell envelope represents the main line of host defense that viruses encounter on their way from one cell to another. The cytoplasmic membrane in general is a physical barrier that needs to be crossed both upon viral entry and exit. Therefore, viruses from the three domains of life employ a wide range of strategies for perforation of the cell membrane, each adapted to the cell surface environment of their host. Here, we review recent insights on entry and egress mechanisms of viruses infecting archaea. Due to the unique nature of the archaeal cell envelope, these particular viruses exhibit novel and unexpected mechanisms to traverse the cellular membrane. PMID:26097469

  11. A Distinct Role of the Queen in Coordinated Workload and Soil Distribution in Eusocial Naked Mole-Rats

    PubMed Central

    Kutsukake, Nobuyuki; Inada, Masayuki; Sakamoto, Shinsuke H.; Okanoya, Kazuo

    2012-01-01

    We investigated how group members achieve collective decision-making, by considering individual intrinsic behavioural rules and behavioural mechanisms for maintaining social integration. Using a simulated burrow environment, we investigated the behavioural rules of coordinated workload for soil distribution in a eusocial mammal, the naked mole-rat (Heterocephalus glaber). We tested two predictions regarding a distinct role of the queen, a socially dominant individual in the caste system: the presence of a queen would increase the workload of other caste individuals, and the cues by a queen would affect the soil distribution. In experiment 1, we placed four individuals of various castes from the same colony into an experimental burrow. Workers exhibited the highest frequency of workload compared to other castes. The presence of a queen activated the workload by other individuals. Individuals showed a consistent workload in a particular direction so as to bias the soil distribution. These results suggest that individuals have a consensus on soil distribution and that the queen plays a distinct role. In experiment 2, we placed the odour of a queen in one of four cells and observed its effect on other individuals’ workload and soil distribution. Relative to other cells, individuals frequently dug in the queen cell so the amount of soil in the queen cell decreased. These results suggest that queen odour is an important cue in coordinated workload and soil distribution in this species. PMID:22957085

  12. Propagation of infectious salmon anaemia (ISA) virus in cell culture.

    PubMed

    Dannevig, B H; Falk, K; Press, C M

    1995-01-01

    A long-term cell line supporting growth of the infectious salmon anaemia (ISA) virus has been established. The cell line (SHK-1) was developed from a culture of head kidney leucocytes from Atlantic salmon, and exhibited macrophage-like enzyme reactivities. By means of transmission experiments, ISA infectivity of cell culture medium could be demonstrated from day 5 after infection of SHK-1 cells with ISA-infective tissue homogenate. ISA infectivity of cell culture medium increased following repeated passages of virus. ISA-infected cell cultures develop cytopathic effects (CPE), making quantitation of virus possible. The development of CPE in ISA virus infected cells was inhibited by ammonium chloride, chloroquine and bafilomycin A, suggesting that infection of SHK-1 cells with ISA virus requires a low-pH step. PMID:8581019

  13. Propagation and Titration of West Nile Virus on Vero Cells.

    PubMed

    McAuley, Alexander J; Beasley, David W C

    2016-01-01

    The propagation and titration of viruses are key virological techniques. Unlike other flaviviruses, such as the dengue viruses, West Nile virus (WNV) grows and plaques very efficiently on Vero cells, usually inducing strong cytopathic effect (CPE) and forming clear plaques. Here, we outline the steps for propagating WNV from culture supernatant stocks and homogenized organ/mosquito samples, as well as for determining virus titers in samples by serial-dilution plaque assay using neutral red or crystal violet stains. PMID:27188547

  14. The use of quantitative PCR to detect Felis catus papillomavirus type 2 DNA from a high proportion of queens and their kittens.

    PubMed

    Thomson, N A; Dunowska, M; Munday, J S

    2015-02-25

    Squamous cell carcinomas are common feline skin cancers that have been associated with infection with Felis catus papillomavirus type 2 (FcaPV-2). Currently, little is known about the epidemiology of FcaPV-2 infection. The aim of this study was to develop a real-time PCR assay to quantify FcaPV-2 DNA in plucked hairs and skin swabs from 11 healthy breeding queens and their kittens. Samples were taken prior to kittening and then 2, 7 and 28 days after kittening to determine the age at which the kittens were first exposed to the virus. FcaPV-2 DNA was amplified from all of the queens and from 91% of the kittens at 2 days of age. There was a wide range in the quantity of FcaPV-2 DNA detected, from 1 to 92,520 copies per swab, and from 0.01 to 234 copies per copy of reference gene DNA in the hair plucks. The quantity of FcaPV-2 DNA detected in samples collected from the kittens was strongly correlated to that of their respective queens and the mean viral DNA load was similar for cats within a household but varied significantly between households. This is the first time that quantitative PCR has been used to detect FcaPV-2 DNA and the results suggest that the virus is ubiquitous but there is a wide variation of viral DNA loads. Kittens appear to be exposed to FcaPV-2 early in life, presumably from direct contact with their queen. These results are important when determining if FcaPV-2 infection of cats is preventable. PMID:25541379

  15. Agglutination of Sindbis Virus and of Cells Infected with Sindbis Virus by Plant Lectins

    PubMed Central

    Birdwell, Charles R.; Strauss, James H.

    1973-01-01

    We have examined the agglutination of Sindbis virus and of chick and hamster cells infected with Sindbis virus by two of the plant lectins, concanavalin A and Ricinus communis agglutinin. Both lectins agglutinate the virus by binding to the polysaccharide chains of the envelope glycoproteins. Both chick and hamster cells exhibit increased agglutination by the lectins after infection by Sindbis virus. In the case of chick cells infected with Sindbis virus, this increase in agglutinability occurs between 3 and 5 h after infection. Infected and mock-infected cells bind the same amount of 3H-labeled concanavalin A, which suggests that the increase in agglutination after infection is due to rearrangements at the cell surface rather than to insertion of new lectin binding sites per se. PMID:4735591

  16. Permissive and restricted virus infection of murine embryonic stem cells.

    PubMed

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey; Kellam, Paul

    2012-10-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA 'off-target' effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host-pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host-virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host-virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  17. Permissive and restricted virus infection of murine embryonic stem cells

    PubMed Central

    Wash, Rachael; Calabressi, Sabrina; Franz, Stephanie; Griffiths, Samantha J.; Goulding, David; Tan, E-Pien; Wise, Helen; Digard, Paul; Haas, Jürgen; Efstathiou, Stacey

    2012-01-01

    Recent RNA interference (RNAi) studies have identified many host proteins that modulate virus infection, but small interfering RNA ‘off-target’ effects and the use of transformed cell lines limit their conclusiveness. As murine embryonic stem (mES) cells can be genetically modified and resources exist where many and eventually all known mouse genes are insertionally inactivated, it was reasoned that mES cells would provide a useful alternative to RNAi screens. Beyond allowing investigation of host–pathogen interactions in vitro, mES cells have the potential to differentiate into other primary cell types, as well as being used to generate knockout mice for in vivo studies. However, mES cells are poorly characterized for virus infection. To investigate whether ES cells can be used to explore host–virus interactions, this study characterized the responses of mES cells following infection by herpes simplex virus type 1 (HSV-1) and influenza A virus. HSV-1 replicated lytically in mES cells, although mES cells were less permissive than most other cell types tested. Influenza virus was able to enter mES cells and express some viral proteins, but the replication cycle was incomplete and no infectious virus was produced. Knockdown of the host protein AHCYL1 in mES cells reduced HSV-1 replication, showing the potential for using mES cells to study host–virus interactions. Transcriptional profiling, however, indicated the lack of an efficient innate immune response in these cells. mES cells may thus be useful to identify host proteins that play a role in virus replication, but they are not suitable to determine factors that are involved in innate host defence. PMID:22815272

  18. Human Muscle Satellite Cells as Targets of Chikungunya Virus Infection

    PubMed Central

    Ozden, Simona; Huerre, Michel; Riviere, Jean-Pierre; Coffey, Lark L.; Afonso, Philippe V.; Mouly, Vincent; de Monredon, Jean; Roger, Jean-Christophe; El Amrani, Mohamed; Yvin, Jean-Luc; Jaffar, Marie-Christine; Frenkiel, Marie-Pascale; Sourisseau, Marion; Schwartz, Olivier; Butler-Browne, Gillian; Desprès, Philippe; Gessain, Antoine; Ceccaldi, Pierre-Emmanuel

    2007-01-01

    Background Chikungunya (CHIK) virus is a mosquito-transmitted alphavirus that causes in humans an acute infection characterised by fever, polyarthralgia, head-ache, and myalgia. Since 2005, the emergence of CHIK virus was associated with an unprecedented magnitude outbreak of CHIK disease in the Indian Ocean. Clinically, this outbreak was characterized by invalidating poly-arthralgia, with myalgia being reported in 97.7% of cases. Since the cellular targets of CHIK virus in humans are unknown, we studied the pathogenic events and targets of CHIK infection in skeletal muscle. Methodology/Principal Findings Immunohistology on muscle biopsies from two CHIK virus-infected patients with myositic syndrome showed that viral antigens were found exclusively inside skeletal muscle progenitor cells (designed as satelllite cells), and not in muscle fibers. To evaluate the ability of CHIK virus to replicate in human satellite cells, we assessed virus infection on primary human muscle cells; viral growth was observed in CHIK virus-infected satellite cells with a cytopathic effect, whereas myotubes were essentially refractory to infection. Conclusions/Significance This report provides new insights into CHIK virus pathogenesis, since it is the first to identify a cellular target of CHIK virus in humans and to report a selective infection of muscle satellite cells by a viral agent in humans. PMID:17565380

  19. High Genetic Stability of Dengue Virus Propagated in MRC-5 Cells as Compared to the Virus Propagated in Vero Cells

    PubMed Central

    Butler, Michael; Wu, Suh-Chin

    2008-01-01

    This work investigated the replication kinetics of the four dengue virus serotypes (DEN-1 to DEN-4), including dengue virus type 4 (DEN-4) recovered from an infectious cDNA clone, in Vero cells and in MRC-5 cells grown on Cytodex 1 microcarriers. DEN-1 strain Hawaii, DEN-2 strain NGC, DEN-3 strain H-87, and DEN-4 strain H-241 , and DEN-4 strain 814669 derived from cloned DNA, were used to infect Vero cells and MRC-5 cells grown in serum-free or serum-containing microcarrier cultures. Serum-free and serum-containing cultures were found to yield comparable titers of these viruses. The cloned DNA-derived DEN-4 started genetically more homogeneous was used to investigate the genetic stability of the virus propagated in Vero cells and MRC-5 cells. Sequence analysis revealed that the DEN-4 propagated in MRC-5 cells maintained a high genetic stability, compared to the virus propagated in Vero cells. Amino acid substitutions of Gly104Cys and Phe108Ile were detected at 70%, 60%, respectively, in the envelope (E) protein of DEN-4 propagated in Vero cells, whereas a single mutation of Glu345Lys was detected at 50% in E of the virus propagated in MRC-5 cells. Sequencing of multiple clones of three separate DNA fragments spanning 40% of the genome also indicated that DEN-4 propagated in Vero cells contained a higher number of mutations than the virus growing in MRC-5 cells. Although Vero cells yielded a peak virus titer approximately 1 to 17 folds higher than MRC-5 cells, cloned DEN-4 from MRC-5 cells maintained a greater stability than the virus from Vero cells. Serum-free microcarrier cultures of MRC-5 cells offer a potentially valuable system for the large-scale production of live-attenuated DEN vaccines. PMID:18350148

  20. Bluetongue virus mammalian cell surface receptors: Role of glycosaminologycans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Binding and infection rates of bluetongue virus (BTV) on glycosaminoglycan (GAG) and glucosaminoglycan deficient and wild type CHO cell lines and bovine pulmonary artery endothelial cells were determined in the presence or absence of GAG and sialic acid antagonists. Data showed that virus binding ...

  1. Beyond the Black Queen Hypothesis.

    PubMed

    Mas, Alix; Jamshidi, Shahrad; Lagadeuc, Yvan; Eveillard, Damien; Vandenkoornhuyse, Philippe

    2016-09-01

    The Black Queen Hypothesis, recently proposed to explain an evolution of dependency based on gene loss, is gaining ground. This paper focuses on how the evolution of dependency transforms interactions and the community. Using agent-based modeling we suggest that species specializing in the consumption of a common good escape competition and therefore favor coexistence. This evolutionary trajectory could open the way for novel long-lasting interactions and a need to revisit the classically accepted assembly rules. Such evolutionary events also reshape the structure and dynamics of communities, depending on the spatial heterogeneity of the common good production. Let Black be the new black! PMID:26953598

  2. Understanding How Zika Virus Enters and Infects Neural Target Cells.

    PubMed

    Miner, Jonathan J; Diamond, Michael S

    2016-05-01

    Zika virus is a mosquito-transmitted flavivirus that has become a public health concern because of its ability to cause microcephaly. In this issue of Cell Stem Cell, Tang et al. (2016) and Nowakowski et al. (2016) use human neural stem cell models and single-cell RNA sequencing to investigate Zika virus tropism and potential entry receptors. PMID:27152436

  3. Direct assessment of queen quality and lack of worker suppression in a paper wasp

    PubMed Central

    Liebig, Jürgen; Monnin, Thibaud; Turillazzi, Stefano

    2005-01-01

    Assessing a conspecific's potential is often crucial to increase one's fitness, e.g. in female choice, contests with rivals or reproductive conflicts in animal societies. In the latter, helpers benefit from accurately assessing the fertility of the breeder as an indication of inclusive fitness. There is evidence that this can be achieved using chemical correlates of reproductive activity. Here, we show that queen quality can be assessed by directly monitoring her reproductive output. In the paper wasp Polistes dominulus, we mimicked a decrease in queen fertility by regularly removing brood. This triggered ovarian development and egg-laying by many workers, which strongly suggests that brood abundance is a reliable cue of queen quality. Brood abundance can be monitored when workers perform regular brood care in small size societies where each brood element is kept in a separate cell. Our results also show that although the queen was not manipulated, and thus remained healthy and fully fertile, she did not control worker egg-laying. Nevertheless, when workers laid eggs, the queen secured a near reproductive monopoly by selectively destroying these eggs, a mechanism known as ‘queen policing’. By contrast, workers destroyed comparatively few queen-laid eggs, but did destroy each other's eggs. PMID:16006333

  4. Interaction of Human Tumor Viruses with Host Cell Surface Receptors and Cell Entry

    PubMed Central

    Schäfer, Georgia; Blumenthal, Melissa J.; Katz, Arieh A.

    2015-01-01

    Currently, seven viruses, namely Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpes virus (KSHV), high-risk human papillomaviruses (HPVs), Merkel cell polyomavirus (MCPyV), hepatitis B virus (HBV), hepatitis C virus (HCV) and human T cell lymphotropic virus type 1 (HTLV-1), have been described to be consistently associated with different types of human cancer. These oncogenic viruses belong to distinct viral families, display diverse cell tropism and cause different malignancies. A key to their pathogenicity is attachment to the host cell and entry in order to replicate and complete their life cycle. Interaction with the host cell during viral entry is characterized by a sequence of events, involving viral envelope and/or capsid molecules as well as cellular entry factors that are critical in target cell recognition, thereby determining cell tropism. Most oncogenic viruses initially attach to cell surface heparan sulfate proteoglycans, followed by conformational change and transfer of the viral particle to secondary high-affinity cell- and virus-specific receptors. This review summarizes the current knowledge of the host cell surface factors and molecular mechanisms underlying oncogenic virus binding and uptake by their cognate host cell(s) with the aim to provide a concise overview of potential target molecules for prevention and/or treatment of oncogenic virus infection. PMID:26008702

  5. The ancient Virus World and evolution of cells

    PubMed Central

    Koonin, Eugene V; Senkevich, Tatiana G; Dolja, Valerian V

    2006-01-01

    Background Recent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones. Results Several genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of

  6. Targeting Cancer-initiating Cells With Oncolytic Viruses

    PubMed Central

    Cripe, Timothy P; Wang, Pin-Yi; Marcato, Paola; Mahller, Yonatan Y; Lee, Patrick WK

    2009-01-01

    Recent studies in a variety of leukemias and solid tumors indicate that there is significant heterogeneity with respect to tumor-forming ability within a given population of tumor cells, suggesting that only a subpopulation of cells is responsible for tumorigenesis. These cells have been commonly referred to as cancer stem cells (CSCs) or cancer-initiating cells (CICs). CICs have been shown to be relatively resistant to conventional anticancer therapies and are thus thought to be responsible for disease relapse. As such, they represent a potentially critical therapeutic target. Oncolytic viruses are in clinical trials for cancer and kill cells through mechanisms different from conventional therapeutics. Because these viruses are not susceptible to the same pathways of drug or radiation resistance, it is important to learn whether CICs are susceptible to oncolytic virus infection. Here we review the available data regarding the ability of several different oncolytic virus types to target CICs for destruction. PMID:19672244

  7. In vivo and in vitro infection dynamics of honey bee viruses.

    PubMed

    Carrillo-Tripp, Jimena; Dolezal, Adam G; Goblirsch, Michael J; Miller, W Allen; Toth, Amy L; Bonning, Bryony C

    2016-01-01

    The honey bee (Apis mellifera) is commonly infected by multiple viruses. We developed an experimental system for the study of such mixed viral infections in newly emerged honey bees and in the cell line AmE-711, derived from honey bee embryos. When inoculating a mixture of iflavirids [sacbrood bee virus (SBV), deformed wing virus (DWV)] and dicistrovirids [Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV)] in both live bee and cell culture assays, IAPV replicated to higher levels than other viruses despite the fact that SBV was the major component of the inoculum mixture. When a different virus mix composed mainly of the dicistrovirid Kashmir bee virus (KBV) was tested in cell culture, the outcome was a rapid increase in KBV but not IAPV. We also sequenced the complete genome of an isolate of DWV that covertly infects the AmE-711 cell line, and found that this virus does not prevent IAPV and KBV from accumulating to high levels and causing cytopathic effects. These results indicate that different mechanisms of virus-host interaction affect virus dynamics, including complex virus-virus interactions, superinfections, specific virus saturation limits in cells and virus specialization for different cell types. PMID:26923109

  8. In vivo and in vitro infection dynamics of honey bee viruses

    PubMed Central

    Carrillo-Tripp, Jimena; Dolezal, Adam G.; Goblirsch, Michael J.; Miller, W. Allen; Toth, Amy L.; Bonning, Bryony C.

    2016-01-01

    The honey bee (Apis mellifera) is commonly infected by multiple viruses. We developed an experimental system for the study of such mixed viral infections in newly emerged honey bees and in the cell line AmE-711, derived from honey bee embryos. When inoculating a mixture of iflavirids [sacbrood bee virus (SBV), deformed wing virus (DWV)] and dicistrovirids [Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV)] in both live bee and cell culture assays, IAPV replicated to higher levels than other viruses despite the fact that SBV was the major component of the inoculum mixture. When a different virus mix composed mainly of the dicistrovirid Kashmir bee virus (KBV) was tested in cell culture, the outcome was a rapid increase in KBV but not IAPV. We also sequenced the complete genome of an isolate of DWV that covertly infects the AmE-711 cell line, and found that this virus does not prevent IAPV and KBV from accumulating to high levels and causing cytopathic effects. These results indicate that different mechanisms of virus-host interaction affect virus dynamics, including complex virus-virus interactions, superinfections, specific virus saturation limits in cells and virus specialization for different cell types. PMID:26923109

  9. Early Events in Chikungunya Virus Infection—From Virus Cell Binding to Membrane Fusion

    PubMed Central

    van Duijl-Richter, Mareike K. S.; Hoornweg, Tabitha E.; Rodenhuis-Zybert, Izabela A.; Smit, Jolanda M.

    2015-01-01

    Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne alphavirus causing millions of infections in the tropical and subtropical regions of the world. CHIKV infection often leads to an acute self-limited febrile illness with debilitating myalgia and arthralgia. A potential long-term complication of CHIKV infection is severe joint pain, which can last for months to years. There are no vaccines or specific therapeutics available to prevent or treat infection. This review describes the critical steps in CHIKV cell entry. We summarize the latest studies on the virus-cell tropism, virus-receptor binding, internalization, membrane fusion and review the molecules and compounds that have been described to interfere with virus cell entry. The aim of the review is to give the reader a state-of-the-art overview on CHIKV cell entry and to provide an outlook on potential new avenues in CHIKV research. PMID:26198242

  10. Antibody secreting cell assay for influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An ELISPOT assay to enumerate B-cells producing antibodies specific to a given antigen, also known as an antibody secreting cell (ASC) assay, was adapted to detect B-cells specific for influenza A virus (IAV). The assay is performed ex vivo and enumerates ASC at a single cell level. A simple ASC det...

  11. Restricted Replication of Vesicular Stomatitis Virus in Human Lymphoblastoid Cells

    PubMed Central

    Nowakowski, Maja; Bloom, Barry R.; Ehrenfeld, Ellie; Summers, Donald F.

    1973-01-01

    Replication of vesicular stomatitis virus (VSV) is restricted in one human lymphoblastoid cell line (Raji), but not in another similar cell line (Wil-2), compared with growth in HeLa cells. This restriction is characterized by a low proportion of cells yielding infectious virus and is associated with limited production of 42S virion RNA. Primary transcription of 13S and 26S VSV-specific RNA is not restricted in Raji cells, and the 13S RNA produced contains adenylate-rich sequences. This suggests that the block in Raji cells involves some step required for the replication of virion RNA. PMID:4357508

  12. Diagnostic approaches for viruses and prions in stem cell banks

    SciTech Connect

    Cobo, Fernando . E-mail: fernancobo@fundacionhvn.org; Talavera, Paloma; Concha, Angel

    2006-03-30

    Some stem cell lines may contain an endogenous virus or can be contaminated with exogenous viruses (even of animal origin) and may secrete viral particles or express viral antigens on their surface. Moreover, certain biotechnological products (e.g. bovine fetal serum, murine feeder cells) may contain prion particles. Viral and prion contamination of cell cultures and 'feeder' cells, which is a common risk in all biotechnological products derived from the cell lines, is the most challenging and potentially serious outcome to address, due to the difficulty involved in virus and prion detection and the potential to cause serious disease in recipients of these cell products. Stem cell banks should introduce adequate quality assurance programs like the microbiological control program and can provide researchers with valuable support in the standardization and safety of procedures and protocols used for the viral and prion testing and in validation programs to assure the quality and safety of the cells.

  13. Modelling Spread of Oncolytic Viruses in Heterogeneous Cell Populations

    NASA Astrophysics Data System (ADS)

    Ellis, Michael; Dobrovolny, Hana

    2014-03-01

    One of the most promising areas in current cancer research and treatment is the use of viruses to attack cancer cells. A number of oncolytic viruses have been identified to date that possess the ability to destroy or neutralize cancer cells while inflicting minimal damage upon healthy cells. Formulation of predictive models that correctly describe the evolution of infected tumor systems is critical to the successful application of oncolytic virus therapy. A number of different models have been proposed for analysis of the oncolytic virus-infected tumor system, with approaches ranging from traditional coupled differential equations such as the Lotka-Volterra predator-prey models, to contemporary modeling frameworks based on neural networks and cellular automata. Existing models are focused on tumor cells and the effects of virus infection, and offer the potential for improvement by including effects upon normal cells. We have recently extended the traditional framework to a 2-cell model addressing the full cellular system including tumor cells, normal cells, and the impacts of viral infection upon both populations. Analysis of the new framework reveals complex interaction between the populations and potential inability to simultaneously eliminate the virus and tumor populations.

  14. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 50 Wildlife and Fisheries 12 2013-10-01 2013-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  15. 50 CFR 622.493 - Landing Caribbean queen conch intact.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 50 Wildlife and Fisheries 12 2014-10-01 2014-10-01 false Landing Caribbean queen conch intact. 622... ATLANTIC Queen Conch Resources of Puerto Rico and the U.S. Virgin Islands § 622.493 Landing Caribbean queen conch intact. (a) A Caribbean queen conch in or from the Caribbean EEZ must be maintained with meat...

  16. Monitoring Physiological Changes in Haloarchaeal Cell during Virus Release

    PubMed Central

    Svirskaitė, Julija; Oksanen, Hanna M.; Daugelavičius, Rimantas; Bamford, Dennis H.

    2016-01-01

    The slow rate of adsorption and non-synchronous release of some archaeal viruses have hindered more thorough analyses of the mechanisms of archaeal virus release. To address this deficit, we utilized four viruses that infect Haloarcula hispanica that represent the four virion morphotypes currently known for halophilic euryarchaeal viruses: (1) icosahedral internal membrane-containing SH1; (2) icosahedral tailed HHTV-1; (3) spindle-shaped His1; and (4) pleomorphic His2. To discern the events occurring as the progeny viruses exit, we monitored culture turbidity, as well as viable cell and progeny virus counts of infected and uninfected cultures. In addition to these traditional metrics, we measured three parameters associated with membrane integrity: the binding of the lipophilic anion phenyldicarbaundecaborane, oxygen consumption, and both intra- and extra-cellular ATP levels. PMID:26927156

  17. Ultrastructure of Zika virus particles in cell cultures.

    PubMed

    Barreto-Vieira, Debora Ferreira; Barth, Ortrud Monika; Silva, Marcos Alexandre Nunes da; Santos, Carolina Cardoso; Santos, Aline da Silva; F, Joaquim Batista; Filippis, Ana Maria Bispo de

    2016-08-01

    Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient's blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible. PMID:27581122

  18. Ultrastructure of Zika virus particles in cell cultures

    PubMed Central

    Barreto-Vieira, Debora Ferreira; Barth, Ortrud Monika; da Silva, Marcos Alexandre Nunes; Santos, Carolina Cardoso; Santos, Aline da Silva; F, Joaquim Batista; de Filippis, Ana Maria Bispo

    2016-01-01

    Zika virus (ZIKV) has infected thousands of Brazilian people and spread to other American countries since 2015. The introduction of ZIKV brought a strong impact to public health in Brazil. It is of utmost importance to identify a susceptible cell line that will enable the isolation and identification of the virus from patient samples, viral mass production, and testing of drug and vaccine candidates. Besides real-time reverse transcriptase polymerase chain reaction diagnosis for detecting the viral genome, virus isolation in cell lines was useful in order to study the structure of the viral particle and its behaviour inside cells. Analysis of ZIKV infected cell lines was achieved using transmission electron microscopy (TEM). Blood was obtained from a Brazilian patient during the first days after presenting with signs of the disease, and ZIKV from the patient’s blood was isolated in the C6/36 mosquito cell line. Afterwards, Vero cells were inoculated with the viral suspension, fixed six days after inoculation, embedded in polymers, and ultra-thin cut. Like dengue viruses, this flavivirus showed numerous virus particles present inside cellular vesicles thereby confirming the susceptibility of the Vero cell line to ZIKV replication. TEM is a unique technique available to make the virus visible. PMID:27581122

  19. Photosensitization of cultured cells and viruses by pyrene lipids.

    PubMed

    Gatt, S; Dinur, T; Abou-Rabia, S; Kotler, M; Fibach, E

    1990-12-01

    Administration of pyrene-linked fatty acids and lipids to cultured cells or an enveloped (vesicular stomatitis) virus induced photosensitization which, following irradiation with a long ultra-violet light (LUV), resulted in killing of the cells and loss of the infectivity of the virus with the following specific effects. (i) LUV illumination of the pyrene-sphingomyelin administered cultured skin fibroblasts derived from normal individuals and patients with Niemann-Pick disease permitted selective killing of the latter. (ii) Similarly LUV illumination of pyrenedodecanoic acid (P12) incubates of leukemic cell lines mixed with human bone marrow cells permitted selective killing of the former. (iii) LUV illumination of P12 incubates of vesicular stomatitis virus decreased the infectivity of the virus by up to 12 logs. PMID:1966337

  20. First study of different insect cells to triatoma virus infection.

    PubMed

    Susevich, María Laura; Marti, Gerardo Aníbal; Metz, Germán Ernesto; Echeverría, María Gabriela

    2015-04-01

    The use of viruses for biological control is a new option to be considered. The family Dicistroviridae, which affects only invertebrates, is one of the families that have been proposed for this purpose. The Triatoma virus (TrV), a member of this family, affects triatomine transmitters of Chagas disease, which is endemic in Latin America but also expanding its worldwide distribution. To this end, we attempted virus replication in Diptera, Aedes albopictus (clone C6/36) and Lepidoptera Spodoptera frugiperda (SF9, SF21) and High Five (H5) cell lines. The methodologies used were transfection process, direct inoculation (purified virus), and inoculation of purified virus with trypsin. Results were confirmed by SDS-PAGE, Western blotting, RT-PCR, electron microscopy, and immunofluorescence. According to the results obtained, further analysis of susceptibility/infection of H5 cells to TrV required to be studied. PMID:25481388

  1. Hepatitis C virus cell-cell transmission and resistance to direct-acting antiviral agents.

    PubMed

    Xiao, Fei; Fofana, Isabel; Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H; Zeisel, Mirjam B; Baumert, Thomas F

    2014-05-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

  2. Hepatitis C Virus Cell-Cell Transmission and Resistance to Direct-Acting Antiviral Agents

    PubMed Central

    Heydmann, Laura; Barth, Heidi; Soulier, Eric; Habersetzer, François; Doffoël, Michel; Bukh, Jens; Patel, Arvind H.; Zeisel, Mirjam B.; Baumert, Thomas F.

    2014-01-01

    Hepatitis C virus (HCV) is transmitted between hepatocytes via classical cell entry but also uses direct cell-cell transfer to infect neighboring hepatocytes. Viral cell-cell transmission has been shown to play an important role in viral persistence allowing evasion from neutralizing antibodies. In contrast, the role of HCV cell-cell transmission for antiviral resistance is unknown. Aiming to address this question we investigated the phenotype of HCV strains exhibiting resistance to direct-acting antivirals (DAAs) in state-of-the-art model systems for cell-cell transmission and spread. Using HCV genotype 2 as a model virus, we show that cell-cell transmission is the main route of viral spread of DAA-resistant HCV. Cell-cell transmission of DAA-resistant viruses results in viral persistence and thus hampers viral eradication. We also show that blocking cell-cell transmission using host-targeting entry inhibitors (HTEIs) was highly effective in inhibiting viral dissemination of resistant genotype 2 viruses. Combining HTEIs with DAAs prevented antiviral resistance and led to rapid elimination of the virus in cell culture model. In conclusion, our work provides evidence that cell-cell transmission plays an important role in dissemination and maintenance of resistant variants in cell culture models. Blocking virus cell-cell transmission prevents emergence of drug resistance in persistent viral infection including resistance to HCV DAAs. PMID:24830295

  3. [Culture and control of cells producing bovine leukemia virus].

    PubMed

    Granátová, M

    1987-10-01

    In the field surveys of the occurrence of enzootic bovine leucosis caused by the bovine leucosis virus (BLV), the identification of positive animals is based on the detection of specific antiviral antibodies by serological methods. The reliability of these tests (particularly their sensitivity and specificity) depends on the quality of the virus antigen. The preparation of the antigen is based on the cultivation of BLV virus in cultures of the FLS cell line. A modified procedure of preparing the BLV antigen in the FLS cell culture is described, along with the control of its production by the immunoperoxidase test. PMID:2827363

  4. Antitumor efficacy of vaccinia virus-modified tumor cell vaccine

    SciTech Connect

    Ito, T.; Wang, D.Q.; Maru, M.; Nakajima, K.; Kato, S.; Kurimura, T.; Wakamiya, N. )

    1990-11-01

    The antitumor efficacies of vaccinia virus-modified tumor cell vaccines were examined in murine syngeneic MH134 and X5563 tumor cells. UV-inactivated vaccinia virus was inoculated i.p. into C3H/HeN mice that had received whole body X-irradiation at 150 rads. After 3 weeks, the vaccines were administered i.p. 3 times at weekly intervals. One week after the last injection, mice were challenged i.p. with various doses of syngeneic MH134 or X5563 viable tumor cells. Four methods were used for preparing tumor cell vaccines: X-ray irradiation; fixation with paraformaldehyde for 1 h or 3 months; and purification of the membrane fraction. All four vaccines were effective, but the former two vaccines were the most effective. A mixture of the membrane fraction of untreated tumor cells and UV-inactivated vaccinia virus also had an antitumor effect. These results indicate that vaccine with the complete cell structure is the most effective. The membrane fraction of UV-inactivated vaccinia virus-absorbed tumor cells was also effective. UV-inactivated vaccinia virus can react with not only intact tumor cells but also the purified membrane fraction of tumor cells and augment antitumor activity.

  5. Dengue Virus Infection Perturbs Lipid Homeostasis in Infected Mosquito Cells

    SciTech Connect

    Perera, Rushika M.; Riley, Catherine; Isaac, Georgis; Hopf- Jannasch, Amber; Moore, Ronald J.; Weitz, Karl K.; Pasa-Tolic, Ljiljana; Metz, Thomas O.; Adamec, Jiri; Kuhn, Richard J.

    2012-03-22

    Dengue virus causes {approx}50-100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture.

  6. Infection of cells by Sindbis virus at low temperature

    SciTech Connect

    Wang Gongbo; Hernandez, Raquel; Weninger, Keith; Brown, Dennis T. . E-mail: dennis_brown@ncsu.edu

    2007-06-05

    Sindbis virus, which belongs to the family Togaviridae genus Alphavirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for Alphaviruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This virus-host membrane fusion model, well established by influenza virus, has been applied to other unrelated membrane-containing viruses including Alphaviruses. The other mechanism proposes direct penetration of the cell plasma membrane by the virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311-3320] studies. We have examined the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis virus infects cells in a temperature- and time-dependent fashion.

  7. Infection of cells by Sindbis virus at low temperature.

    PubMed

    Wang, Gongbo; Hernandez, Raquel; Weninger, Keith; Brown, Dennis T

    2007-06-01

    Sindbis virus, which belongs to the family Togaviridae genus Alphavirus infects a variety of vertebrate and invertebrate cells. The initial steps of Sindbis virus infection involve attachment, penetration and uncoating. Two different pathways of infection have been proposed for Alphaviruses. One proposed mechanism involves receptor mediated virion endocytosis followed by membrane fusion triggered by endosome acidification. This virus-host membrane fusion model, well established by influenza virus, has been applied to other unrelated membrane-containing viruses including Alphaviruses. The other mechanism proposes direct penetration of the cell plasma membrane by the virus glycoproteins in the absence of membrane fusion. This alternate model is supported by both ultrastructural [Paredes, A.M., Ferreira, D., Horton, M., Saad, A., Tsuruta, H., Johnston, R., Klimstra, W., Ryman, K., Hernandez, R., Chiu, W., Brown, D.T., 2004. Conformational changes in Sindbis virions resulting from exposure to low pH and interactions with cells suggest that cell penetration may occur at the cell surface in the absence of membrane fusion. Virology 324(2), 373-386] and biochemical [Koschinski, A., Wengler, G., Wengler, G., and Repp, H., 2005. Rare earth ions block the ion pores generated by the class II fusion proteins of alphaviruses and allow analysis of the biological functions of these pores. J. Gen. Virol. 86(Pt. 12), 3311-3320] studies. We have examined the ability of Sindbis virus to infect Baby Hamster Kidney (BHK) cells at temperatures which block endocytosis. We have found that under these conditions Sindbis virus infects cells in a temperature- and time-dependent fashion. PMID:17289103

  8. Role of mink cell focus-inducing virus in leukemias induced by Friend ecotropic virus.

    PubMed Central

    Silver, J

    1984-01-01

    Recombinant viruses have been implicated in the pathogenesis of murine leukemias induced by a variety of long-latency retroviruses. Neonatal mice of several strains were inoculated with Friend ecotropic virus (F-Eco) and analyzed for the presence of mink cell focus-inducing (MCF) virus or DNA restriction enzyme fragments which were specific for Friend MCF virus (F-MCF). MCF virus was detected within 2 weeks of inoculation in NFS /N mice and at about 2 months after inoculation in BALB/c mice. Both of these strains developed erythroblastosis after inoculation with F-Eco. In contrast, MCF virus was not detected in F-Eco-inoculated C57BL mice. These mice were resistant to erythroblastosis but developed lymphoma or myelogenous leukemia or both at about 5 months after inoculation. Thus, although MCF viruses were associated with F-Eco erythroblastosis in NFS /N and BALB/c mice, they were not necessary for F-Eco-induced lymphoid or myeloid leukemias in C57BL mice. To investigate the association between resistance to erythroblastosis and absence of MCF virus, C57BL mice were inoculated with pseudotypic mixtures of F-Eco plus F-MCF; MCF virus replicated well in these mice, but the mice remained resistant to erythroblastosis. Furthermore, in genetic crosses between C57BL and NFS /N or BALB/c, some mice inherited resistance to F-Eco erythroblastosis without inheriting the C57BL resistance to the generation of MCF viruses. These results indicate that C57BL mice carry a gene for resistance to F-Eco erythroblastosis which is distinct from the C57BL genes which interfere with the generation of MCF viruses. Images PMID:6726889

  9. Potential for bluetongue virus persistence in insect cells.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A potential persistent bluetongue virus (BTV) infection of an insect cell line is under investigation. Culicoides sonorensis, a biting midge, is the primary vector in the U.S. for BTV, which infects sheep, cattle, and wild ruminants. A Culicoides cell line (KC cell line) developed at the ABADRL has...

  10. Influenza virus binds its host cell using multiple dynamic interactions

    PubMed Central

    Sieben, Christian; Kappel, Christian; Zhu, Rong; Wozniak, Anna; Rankl, Christian; Hinterdorfer, Peter; Grubmüller, Helmut; Herrmann, Andreas

    2012-01-01

    Influenza virus belongs to a wide range of enveloped viruses. The major spike protein hemagglutinin binds sialic acid residues of glycoproteins and glycolipids with dissociation constants in the millimolar range [Sauter NK, et al. (1992) Biochemistry 31:9609–9621], indicating a multivalent binding mode. Here, we characterized the attachment of influenza virus to host cell receptors using three independent approaches. Optical tweezers and atomic force microscopy-based single-molecule force spectroscopy revealed very low interaction forces. Further, the observation of sequential unbinding events strongly suggests a multivalent binding mode between virus and cell membrane. Molecular dynamics simulations reveal a variety of unbinding pathways that indicate a highly dynamic interaction between HA and its receptor, allowing rationalization of influenza virus–cell binding quantitatively at the molecular level. PMID:22869709

  11. Efficiency of Cell-Free and Cell-Associated Virus in Mucosal Transmission of Human Immunodeficiency Virus Type 1 and Simian Immunodeficiency Virus

    PubMed Central

    Hulot, Sandrine L.; Korioth-Schmitz, Birgit; Gombos, Randi B.; Zheng, Yi; Owuor, Joshua; Lifton, Michelle A.; Ayeni, Christian; Najarian, Robert M.; Yeh, Wendy W.; Asmal, Mohammed; Zamir, Gideon; Letvin, Norman L.

    2013-01-01

    Effective strategies are needed to block mucosal transmission of human immunodeficiency virus type 1 (HIV-1). Here, we address a crucial question in HIV-1 pathogenesis: whether infected donor mononuclear cells or cell-free virus plays the more important role in initiating mucosal infection by HIV-1. This distinction is critical, as effective strategies for blocking cell-free and cell-associated virus transmission may be different. We describe a novel ex vivo model system that utilizes sealed human colonic mucosa explants and demonstrate in both the ex vivo model and in vivo using the rectal challenge model in rhesus monkeys that HIV-1-infected lymphocytes can transmit infection across the mucosa more efficiently than cell-free virus. These findings may have significant implications for our understanding of the pathogenesis of mucosal transmission of HIV-1 and for the development of strategies to prevent HIV-1 transmission. PMID:24109227

  12. Understanding and altering cell tropism of vesicular stomatitis virus

    PubMed Central

    Hastie, Eric; Cataldi, Marcela; Marriott, Ian; Grdzelishvili, Valery Z.

    2013-01-01

    Vesicular stomatitis virus (VSV) is a prototypic nonsegmented negative-strand RNA virus. VSV’s broad cell tropism makes it a popular model virus for many basic research applications. In addition, a lack of preexisting human immunity against VSV, inherent oncotropism and other features make VSV a widely used platform for vaccine and oncolytic vectors. However, VSV’s neurotropism that can result in viral encephalitis in experimental animals needs to be addressed for the use of the virus as a safe vector. Therefore, it is very important to understand the determinants of VSV tropism and develop strategies to alter it. VSV glycoprotein (G) and matrix (M) protein play major roles in its cell tropism. VSV G protein is responsible for VSV broad cell tropism and is often used for pseudotyping other viruses. VSV M affects cell tropism via evasion of antiviral responses, and M mutants can be used to limit cell tropism to cell types defective in interferon signaling. In addition, other VSV proteins and host proteins may function as determinants of VSV cell tropism. Various approaches have been successfully used to alter VSV tropism to benefit basic research and clinically relevant applications. PMID:23796410

  13. Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells

    PubMed Central

    Steinmetz, Nicole F; Cho, Choi-Fong; Ablack, Amber; Lewis, John D; Manchester, Marianne

    2011-01-01

    Aims Vimentin, a type III intermediate filament, is upregulated during epithelial–mesenchymal transition and tumor progression. Vimentin is surface-expressed on cells involved in inflammation; the function remains unknown. We investigated the expression of surface vimentin on cancer cells and evaluated targeting nanoparticles to tumors exploiting vimentin. Materials & methods Cowpea mosaic virus nanoparticles that interact with surface vimentin were used as probes. Tumor homing was tested using the chick chorioallantoic membrane model with human tumor xenografts. Results & discussion Surface vimentin levels varied during cell cycle and among the cell lines tested. Surface vimentin expression correlated with cowpea mosaic virus uptake, underscoring the utility of cowpea mosaic virus to detect invasive cancer cells. Targeting to tumor xenografts was observed; homing was based on the enhanced permeability and retention effect. Our data provide novel insights into the role of surface vimentin in cancer and targeting nanoparticles in vivo. PMID:21385137

  14. Honey Bee (Apis mellifera) Queen Reproductive Potential Affects Queen Mandibular Gland Pheromone Composition and Worker Retinue Response.

    PubMed

    Rangel, Juliana; Böröczky, Katalin; Schal, Coby; Tarpy, David R

    2016-01-01

    Reproductive division of labor is one of the defining traits of honey bees (Apis mellifera), with non-reproductive tasks being performed by workers while a single queen normally monopolizes reproduction. The decentralized organization of a honey bee colony is maintained in large part by a bouquet of queen-produced pheromones, the distribution of which is facilitated by contact among workers throughout the hive. Previous studies have shown that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures of reproductive potential compared to queens raised from older worker larvae. We investigated differences in the chemical composition of the mandibular glands and attractiveness to workers of "high-quality" queens (i.e., raised from first instar worker larvae; more queen-like) and "low-quality" queens (i.e., raised from third instar worker larvae; more worker-like). We characterized the chemical profiles of the mandibular glands of high-quality queens and low-quality queens using GC-MS and used the worker retinue response as a measure of the attractiveness to workers of high-quality queens vs. low-quality queens. We found that queen quality affected the chemical profiles of mandibular gland contents differently across years, showing significant differences in the production of the queen mandibular pheromone ("QMP") components HVA and 9-HDA in 2010, but no significant differences of any glandular compound in 2012. We also found that workers were significantly more attracted to high-quality queens than to low-quality queens in 2012, possibly because of increased attractiveness of their mandibular gland chemical profiles. Our results indicate that the age at which honey bee larvae enter the "queen-specific" developmental pathway influences the chemical composition of queen mandibular glands and worker behavior. However, these changes are not consistent across years, suggesting that other external

  15. Honey Bee (Apis mellifera) Queen Reproductive Potential Affects Queen Mandibular Gland Pheromone Composition and Worker Retinue Response

    PubMed Central

    Böröczky, Katalin; Schal, Coby; Tarpy, David R.

    2016-01-01

    Reproductive division of labor is one of the defining traits of honey bees (Apis mellifera), with non-reproductive tasks being performed by workers while a single queen normally monopolizes reproduction. The decentralized organization of a honey bee colony is maintained in large part by a bouquet of queen-produced pheromones, the distribution of which is facilitated by contact among workers throughout the hive. Previous studies have shown that the developmental fate of honey bee queens is highly plastic, with queens raised from younger worker larvae exhibiting higher measures of reproductive potential compared to queens raised from older worker larvae. We investigated differences in the chemical composition of the mandibular glands and attractiveness to workers of “high-quality” queens (i.e., raised from first instar worker larvae; more queen-like) and “low-quality” queens (i.e., raised from third instar worker larvae; more worker-like). We characterized the chemical profiles of the mandibular glands of high-quality queens and low-quality queens using GC-MS and used the worker retinue response as a measure of the attractiveness to workers of high-quality queens vs. low-quality queens. We found that queen quality affected the chemical profiles of mandibular gland contents differently across years, showing significant differences in the production of the queen mandibular pheromone (“QMP”) components HVA and 9-HDA in 2010, but no significant differences of any glandular compound in 2012. We also found that workers were significantly more attracted to high-quality queens than to low-quality queens in 2012, possibly because of increased attractiveness of their mandibular gland chemical profiles. Our results indicate that the age at which honey bee larvae enter the “queen-specific” developmental pathway influences the chemical composition of queen mandibular glands and worker behavior. However, these changes are not consistent across years, suggesting

  16. Structure and cell biology of archaeal virus STIV.

    PubMed

    Fu, Chi-yu; Johnson, Johnson E

    2012-04-01

    Recent investigations of archaeal viruses have revealed novel features of their structures and life cycles when compared to eukaryotic and bacterial viruses, yet there are structure-based unifying themes suggesting common ancestral relationships among dsDNA viruses in the three kingdoms of life. Sulfolobus solfataricus and the infecting virus Sulfolobus turreted icosahedral virus (STIV) is one of the well-established model systems to study archaeal virus replication and viral-host interactions. Reliable laboratory conditions to propagate STIV and available genetic tools allowed structural characterization of the virus and viral components that lead to the proposal of common capsid ancestry with PRD1 (bacteriophage), Adenovirus (eukaryotic virus) and PBCV (chlorellavirus). Microarray and proteomics approaches systematically analyzed viral replication and the corresponding host responses. Cellular cryo-electron tomography and thin-section EM studies uncovered the assembly and maturation pathway of STIV and revealed dramatic cellular ultra-structure changes upon infection. The viral-induced pyramid-like protrusions on cell surfaces represent a novel viral release mechanism and previously uncharacterized functions in viral replication. PMID:22482708

  17. Induction of apoptosis in frog virus 3-infected cells.

    PubMed

    Chinchar, V G; Bryan, Locke; Wang, J; Long, Scott; Chinchar, G D

    2003-02-15

    The ability of frog virus 3 (FV3), the type species of the family Iridoviridae, to induce apoptosis was examined by monitoring DNA cleavage, chromatin condensation, and cell-surface expression of phosphotidylserine (PS) in fathead minnow (FHM) and baby hamster kidney (BHK) cells. In productively infected FHM cells, DNA fragmentation was first noted at 6-7 h postinfection and was clearly seen by 17 h postinfection, while chromatin condensation was detected at 8.5 h postinfection. As with some other viruses, FV3-induced apoptosis did not require de novo viral gene expression as both heat-inactivated and UV-inactivated virus readily triggered DNA fragmentation in FHM cells. Moreover, FV3-induced apoptosis was blocked in FHM cells by the pan-caspase inhibitor Z-VAD-FMK, suggesting that virus infection triggers programmed cell death through activation of the caspase cascade. FV3 infection also triggered apoptosis in BHK cells as monitored by TUNEL and annexin V binding assays. To determine whether FV3, similar to other large DNA viruses, encoded proteins that block or delay apoptosis, mock- and FV3-infected FHM cells were osmotically shocked and assayed for DNA fragmentation 3 hours later. DNA fragmentation was clearly seen whether or not shocked cells were previously infected with FV3, indicating that infection with FV3 did not block apoptosis induced by osmotic shock in FHM cells. The above results demonstrate that iridoviruses triggered apoptosis and that the induction of programmed cell death did not require viral gene expression. However, it remains to be determined if virion attachment to target cells is sufficient to induce cell death, or if apoptosis is triggered directly or indirectly by one or more virion-associated proteins. PMID:12642103

  18. Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses.

    PubMed

    Cheng, Li-Lin; Luk, Yan-Yeung; Murphy, Christopher J; Israel, Barbara A; Abbott, Nicholas L

    2005-12-01

    We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using

  19. Measles virus breaks through epithelial cell barriers to achieve transmission

    PubMed Central

    Takeda, Makoto

    2008-01-01

    Measles is a highly contagious disease that causes immunosuppression in patients. Measles virus infection has been thought to begin in the respiratory epithelium and then spread to lymphoid tissue. In this issue of the JCI, Leonard et al. provide data to suggest an alternative model of measles virus pathogenesis (see the related article beginning on page 2448). In human primary epithelial cells and rhesus monkeys in vivo, the authors show that initial infection of respiratory epithelium is not necessary for the virus to enter the host but that viral entry into epithelial cells via interaction of the virus with a receptor located on the basolateral side of the epithelium is required for viral shedding into the airway and subsequent transmission. PMID:18568081

  20. Tinkering with Translation: Protein Synthesis in Virus-Infected Cells

    PubMed Central

    Walsh, Derek; Mathews, Michael B.; Mohr, Ian

    2013-01-01

    Viruses are obligate intracellular parasites, and their replication requires host cell functions. Although the size, composition, complexity, and functions encoded by their genomes are remarkably diverse, all viruses rely absolutely on the protein synthesis machinery of their host cells. Lacking their own translational apparatus, they must recruit cellular ribosomes in order to translate viral mRNAs and produce the protein products required for their replication. In addition, there are other constraints on viral protein production. Crucially, host innate defenses and stress responses capable of inactivating the translation machinery must be effectively neutralized. Furthermore, the limited coding capacity of the viral genome needs to be used optimally. These demands have resulted in complex interactions between virus and host that exploit ostensibly virus-specific mechanisms and, at the same time, illuminate the functioning of the cellular protein synthesis apparatus. PMID:23209131

  1. Global Dynamics of a Virus Dynamical Model with Cell-to-Cell Transmission and Cure Rate

    PubMed Central

    Zhang, Tongqian; Meng, Xinzhu; Zhang, Tonghua

    2015-01-01

    The cure effect of a virus model with both cell-to-cell transmission and cell-to-virus transmission is studied. By the method of next generation matrix, the basic reproduction number is obtained. The locally asymptotic stability of the virus-free equilibrium and the endemic equilibrium is considered by investigating the characteristic equation of the model. The globally asymptotic stability of the virus-free equilibrium is proved by constructing suitable Lyapunov function, and the sufficient condition for the globally asymptotic stability of the endemic equilibrium is obtained by constructing suitable Lyapunov function and using LaSalle invariance principal. PMID:26504489

  2. Virus and Host Mechanics Support Membrane Penetration and Cell Entry.

    PubMed

    Greber, Urs F

    2016-04-01

    Viruses are quasi-inert macromolecular assemblies. Their metastable conformation changes during entry into cells, when chemical and mechanical host cues expose viral membrane-interacting proteins. This leads to membrane rupture or fusion and genome uncoating. Importantly, virions tune their physical properties and enhance penetration and uncoating. For example, influenza virus softens at low pH to uncoat. The stiffness and pressure of adenovirus control uncoating and membrane penetration. Virus and host mechanics thus present new opportunities for antiviral therapy. PMID:26842477

  3. Measles virus persistence in an immortalized murine macrophage cell line.

    PubMed

    Goldman, M B; Buckthal, D J; Picciotto, S; O'Bryan, T A; Goldman, J N

    1995-02-20

    Persistent infection with the Edmonston strain of measles virus (MV) has been established in IC-21 cells, an immortalized murine macrophage cell line. Persistence was established immediately without syncytia formation or cytopathic effects. MV was expressed in the majority of the cells as evidenced by immunofluorescence microscopy, flow cytometry, infectious centers assays, and limiting dilution analysis. Hemagglutinin (H) and phosphoprotein expressed in persistently infected IC-21 cells had retarded migration in SDS-PAGE gels when compared to these proteins expressed in Vero cells. H protein differences were also found between freshly infected IC-21 cells and persistently infected IC-21 cells passaged for over 2 years. Six sublines of IC-21 cells, infected at different times, have maintained these characteristics for 2 years of passage. During this time period the intensity of immunofluorescence and the number of infectious virus particles recoverable fluctuated in five of the six cell lines. In one cell line virus expression remained at a consistent high level. The ability to establish a persistent MV infection in murine macrophages allows studies using a cell important in disseminating the infection. It facilitates experiments on immunological aspects of viral immunity by enabling cell mixing experiments with histocompatible cell populations and by making available the wide array of cellular and humoral reagents in the mouse. PMID:7871720

  4. Viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lytic bacteriophages, viruses which infect and lyse bacterial cells, can provide a natural method to reduce bacterial pathogens on produce commodities. The use of multi-phage cocktails is most likely to be effective against bacterial pathogens on produce commodities, and minimize the development of...

  5. Superior In Vitro Stimulation of Human CD8+ T-Cells by Whole Virus versus Split Virus Influenza Vaccines

    PubMed Central

    Distler, Eva; Dass, Martin; Wagner, Eva M.; Plachter, Bodo; Probst, Hans Christian; Strand, Dennis; Hartwig, Udo F.; Karner, Anita; Aichinger, Gerald; Kistner, Otfried; Landfester, Katharina; Herr, Wolfgang

    2014-01-01

    Pandemic and seasonal influenza viruses cause considerable morbidity and mortality in the general human population. Protection from severe disease may result from vaccines that activate antigen-presenting DC for effective stimulation of influenza-specific memory T cells. Special attention is paid to vaccine-induced CD8+ T-cell responses, because they are mainly directed against conserved internal influenza proteins thereby presumably mediating cross-protection against circulating seasonal as well as emerging pandemic virus strains. Our study showed that influenza whole virus vaccines of major seasonal A and B strains activated DC more efficiently than those of pandemic swine-origin H1N1 and pandemic-like avian H5N1 strains. In contrast, influenza split virus vaccines had a low ability to activate DC, regardless which strain was investigated. We also observed that whole virus vaccines stimulated virus-specific CD8+ memory T cells much stronger compared to split virus counterparts, whereas both vaccine formats activated CD4+ Th cell responses similarly. Moreover, our data showed that whole virus vaccine material is delivered into the cytosolic pathway of DC for effective activation of virus-specific CD8+ T cells. We conclude that vaccines against seasonal and pandemic (-like) influenza strains that aim to stimulate cross-reacting CD8+ T cells should include whole virus rather than split virus formulations. PMID:25072749

  6. Differential effect of p7 inhibitors on hepatitis C virus cell-to-cell transmission☆

    PubMed Central

    Meredith, L.W.; Zitzmann, N.; McKeating, J.A.

    2013-01-01

    Inhibitors targeting the hepatitis C virus (HCV) encoded viroporin, p7 prevent virus release in vitro. HCV can transmit by cell-free particle infection of new target cells and via cell-to-cell dependent contact with limited exposure to the extracellular environment. The role of assembly inhibitors in preventing HCV transmission via these pathways has not been studied. We compared the efficacy of three published p7 inhibitors to inhibit cell-free and cell-to-cell transmission of two chimeric HCV strains encoding genotype 2 (GT2) or 5 (GT5) p7 using a recently developed single cycle co-culture assay. The inhibitors reduced the infectivity of extracellular GT2 and GT5 virus by 80–90% and GT2 virus cell-to-cell transmission by 50%. However, all of the p7 inhibitors had minimal effect on GT5 cell contact dependent transmission. Screening a wider panel of diverse viral genotypes demonstrated that p7 viroporin inhibitors were significantly more effective at blocking cell-free virus than cell-to-cell transmission. These results suggest an altered assembly or trafficking of cell-to-cell transmitted compared to secreted virus. These observations have important implications for the validation, therapeutic design and testing of HCV assembly inhibitors. PMID:24157306

  7. Sensitivity of NCI-H292 human lung mucoepidermoid cells for respiratory and other human viruses.

    PubMed Central

    Hierholzer, J C; Castells, E; Banks, G G; Bryan, J A; McEwen, C T

    1993-01-01

    NCI-H292 mucoepidermoid carcinoma cells from human lungs were shown in an earlier report to be a fully adequate substitute for primary rhesus monkey kidney (MK) cells for the isolation and propagation of the human paramyxoviruses. Although sensitivity for ortho- and paramyxoviruses was the principal reason for using MK cells, the cells were also sensitive to many other viruses, which constituted another important value of MK cells. That MK cells supported the initial isolation and growth of so many respiratory viruses made it a mandatory cell type for any clinical laboratory. We therefore felt it was imperative to evaluate the virus spectrum of NCI-H292 cells, which are being used as a substitute for MK cells in many laboratories. In the present report, we show that NCI-H292 cells are sensitive for vaccinia virus, herpes simplex virus, adenoviruses, BK polyomavirus, reoviruses, measles virus, respiratory syncytial virus, some strains of influenza virus type A, most enteroviruses, and rhinoviruses, in addition to the parainfluenza and mumps viruses originally reported. Furthermore, these viruses replicate in NCI-H292 cells to the same virus and antigen titers and at the same speed of replication as they do in their usually preferred cells. The NCI-H292 cells are therefore an excellent substitute for MK cells in terms of laboratory safety, ease of availability, paramyxovirus isolation, and broad virus spectrum but cannot substitute for MK cells for the isolation of influenza viruses. Images PMID:8314992

  8. Engineering chemically modified viruses for prostate cancer cell recognition.

    PubMed

    Mohan, K; Weiss, G A

    2015-12-01

    Specific detection of circulating tumor cells and characterization of their aggressiveness could improve cancer diagnostics and treatment. Metastasis results from such tumor cells, and causes the majority of cancer deaths. Chemically modified viruses could provide an inexpensive and efficient approach to detect tumor cells and quantitate their cell surface biomarkers. However, non-specific adhesion between the cell surface receptors and the virus surface presents a challenge. This report describes wrapping the virus surface with different PEG architectures, including as fusions to oligolysine, linkers, spacers and scaffolded ligands. The reported PEG wrappers can reduce by >75% the non-specific adhesion of phage to cell surfaces. Dynamic light scattering verified the non-covalent attachment by the reported wrappers as increased sizes of the virus particles. Further modifications resulted in specific detection of prostate cancer cells expressing PSMA, a key prostate cancer biomarker. The approach allowed quantification of PSMA levels on the cell surface, and could distinguish more aggressive forms of the disease. PMID:26463253

  9. "Detection of bluetongue virus in Culicoides cell cultures"

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bluetongue virus (BTV) infects sheep, cattle and other ruminants and is transmitted by Culicoides spp. of biting midges. Cell lines derived from C. sonorensis have been developed at the Arthropod-borne Animal Diseases Research Laboratory. These cell lines have been shown to support BTV replication, ...

  10. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    PubMed

    Malenovská, Hana

    2016-02-01

    Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses. PMID:26562056

  11. Effect of Cell Physiological State on Infection by Rat Virus

    PubMed Central

    Tennant, Raymond W.; Layman, Kenneth R.; Hand, Russell E.

    1969-01-01

    Infection by rat virus has been studied in cultures of rat embryo cells to evaluate the Margolis-Kilham hypothesis that the virus preferentially infects tissues with actively dividing cells. An enhancement of infection was seen in cultures infected 10 hr after fresh medium was added as compared to infection of stationary cultures (infected before addition of fresh medium). Since addition of fresh medium stimulates deoxyribonucleic acid (DNA) synthesis, the number of cells per culture synthesizing DNA at the time of infection was compared with the proportion of cells which synthesized viral protein. Cells were infected before the medium change and 10 or 24 hr after the medium change and were pulse-labeled with 3H-thymidine at the time virus was added. The cells were allowed to initiate viral protein synthesis before they were fixed and stained with fluorescein-conjugated anti-rat virus serum. Fluorescence microscopy permitted both labels to be counted simultaneouly and showed that the greatest proportion of cells synthesizing viral protein were those which had incorporated 3H-thymidine at the time of infection. Images PMID:16789120

  12. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells.

    PubMed

    Villa, Nancy Y; Wasserfall, Clive H; Meacham, Amy M; Wise, Elizabeth; Chan, Winnie; Wingard, John R; McFadden, Grant; Cogle, Christopher R

    2015-06-11

    Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens. PMID:25904246

  13. Myxoma virus suppresses proliferation of activated T lymphocytes yet permits oncolytic virus transfer to cancer cells

    PubMed Central

    Villa, Nancy Y.; Wasserfall, Clive H.; Meacham, Amy M.; Wise, Elizabeth; Chan, Winnie; Wingard, John R.; McFadden, Grant

    2015-01-01

    Allogeneic hematopoietic cell transplant (allo-HCT) can be curative for certain hematologic malignancies, but the risk of graft-versus-host disease (GVHD) is a major limitation for wider application. Ideally, strategies to improve allo-HCT would involve suppression of T lymphocytes that drive GVHD while sparing those that mediate graft-versus-malignancy (GVM). Recently, using a xenograft model, we serendipitously discovered that myxoma virus (MYXV) prevented GVHD while permitting GVM. In this study, we show that MYXV binds to resting, primary human T lymphocytes but will only proceed into active virus infection after the T cells receive activation signals. MYXV-infected T lymphocytes exhibited impaired proliferation after activation with reduced expression of interferon-γ, interleukin-2 (IL-2), and soluble IL-2Rα, but did not affect expression of IL-4 and IL-10. MYXV suppressed T-cell proliferation in 2 patterns (full vs partial) depending on the donor. In terms of GVM, we show that MYXV-infected activated human T lymphocytes effectively deliver live oncolytic virus to human multiple myeloma cells, thus augmenting GVM by transfer of active oncolytic virus to residual cancer cells. Given this dual capacity of reducing GVHD plus increasing the antineoplastic effectiveness of GVM, ex vivo virotherapy with MYXV may be a promising clinical adjunct to allo-HCT regimens. PMID:25904246

  14. Attempts to identify viruses in rheumatoid synovial cells.

    PubMed Central

    Norval, M; Marmion, B P

    1976-01-01

    Synovial fibroblast cell strains derived from the synovial membranes of 7 patients with rheumatoid arthritis were examined for the presence of viruses, in particular leucoviruses. Seven similar synovial strains derived from patients with other arthritic conditions were used as a control group. Evidence of the presence of a virus or a viral genome was looked for by several methods of induction followed by 3H-uridine labelling of the cultures. In addition, the culture supernatant, after induction and after the synovial strains had been co-cultivated with a variety of cell lines from several species, was assayed for the presence of viral RNA-dependent DNA polymerase activity. The DNA-polymerase activity of the synovial cells themselves was also determined. No evidence was found by any of these techniques to indicate the presence of virus or viral information within the synovial fibroblasts. Images PMID:60087

  15. African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

    PubMed Central

    Sánchez, Elena G.; Quintas, Ana; Pérez-Núñez, Daniel; Nogal, Marisa; Barroso, Susana; Carrascosa, Ángel L.; Revilla, Yolanda

    2012-01-01

    African swine fever (ASF) is caused by a large and highly pathogenic DNA virus, African swine fever virus (ASFV), which provokes severe economic losses and expansion threats. Presently, no specific protection or vaccine against ASF is available, despite the high hazard that the continued occurrence of the disease in sub-Saharan Africa, the recent outbreak in the Caucasus in 2007, and the potential dissemination to neighboring countries, represents. Although virus entry is a remarkable target for the development of protection tools, knowledge of the ASFV entry mechanism is still very limited. Whereas early studies have proposed that the virus enters cells through receptor-mediated endocytosis, the specific mechanism used by ASFV remains uncertain. Here we used the ASFV virulent isolate Ba71, adapted to grow in Vero cells (Ba71V), and the virulent strain E70 to demonstrate that entry and internalization of ASFV includes most of the features of macropinocytosis. By a combination of optical and electron microscopy, we show that the virus causes cytoplasm membrane perturbation, blebbing and ruffles. We have also found that internalization of the virions depends on actin reorganization, activity of Na+/H+ exchangers, and signaling events typical of the macropinocytic mechanism of endocytosis. The entry of virus into cells appears to directly stimulate dextran uptake, actin polarization and EGFR, PI3K-Akt, Pak1 and Rac1 activation. Inhibition of these key regulators of macropinocytosis, as well as treatment with the drug EIPA, results in a considerable decrease in ASFV entry and infection. In conclusion, this study identifies for the first time the whole pathway for ASFV entry, including the key cellular factors required for the uptake of the virus and the cell signaling involved. PMID:22719252

  16. Differential Sensitivity of Bat Cells to Infection by Enveloped RNA Viruses: Coronaviruses, Paramyxoviruses, Filoviruses, and Influenza Viruses

    PubMed Central

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  17. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses.

    PubMed

    Hoffmann, Markus; Müller, Marcel Alexander; Drexler, Jan Felix; Glende, Jörg; Erdt, Meike; Gützkow, Tim; Losemann, Christoph; Binger, Tabea; Deng, Hongkui; Schwegmann-Weßels, Christel; Esser, Karl-Heinz; Drosten, Christian; Herrler, Georg

    2013-01-01

    Bats (Chiroptera) host major human pathogenic viruses including corona-, paramyxo, rhabdo- and filoviruses. We analyzed six different cell lines from either Yinpterochiroptera (including African flying foxes and a rhinolophid bat) or Yangochiroptera (genera Carollia and Tadarida) for susceptibility to infection by different enveloped RNA viruses. None of the cells were sensitive to infection by transmissible gastroenteritis virus (TGEV), a porcine coronavirus, or to infection mediated by the Spike (S) protein of SARS-coronavirus (SARS-CoV) incorporated into pseudotypes based on vesicular stomatitis virus (VSV). The resistance to infection was overcome if cells were transfected to express the respective cellular receptor, porcine aminopeptidase N for TGEV or angiotensin-converting enzyme 2 for SARS-CoV. VSV pseudotypes containing the S proteins of two bat SARS-related CoV (Bg08 and Rp3) were unable to infect any of the six tested bat cell lines. By contrast, viral pseudotypes containing the surface protein GP of Marburg virus from the family Filoviridae infected all six cell lines though at different efficiency. Notably, all cells were sensitive to infection by two paramyxoviruses (Sendai virus and bovine respiratory syncytial virus) and three influenza viruses from different subtypes. These results indicate that bat cells are more resistant to infection by coronaviruses than to infection by paramyxoviruses, filoviruses and influenza viruses. Furthermore, these results show a receptor-dependent restriction of the infection of bat cells by CoV. The implications for the isolation of coronaviruses from bats are discussed. PMID:24023659

  18. Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

    PubMed Central

    Kuznetsov, Yurii G.; McPherson, Alexander

    2011-01-01

    Summary: Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM. PMID:21646429

  19. Effects of Insemination Quantity on Honey Bee Queen Physiology

    PubMed Central

    Richard, Freddie-Jeanne; Tarpy, David R.; Grozinger, Christina M.

    2007-01-01

    Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony. PMID

  20. Effects of insemination quantity on honey bee queen physiology.

    PubMed

    Richard, Freddie-Jeanne; Tarpy, David R; Grozinger, Christina M

    2007-01-01

    Mating has profound effects on the physiology and behavior of female insects, and in honey bee (Apis mellifera) queens, these changes are permanent. Queens mate with multiple males during a brief period in their early adult lives, and shortly thereafter they initiate egg-laying. Furthermore, the pheromone profiles of mated queens differ from those of virgins, and these pheromones regulate many different aspects of worker behavior and colony organization. While it is clear that mating causes dramatic changes in queens, it is unclear if mating number has more subtle effects on queen physiology or queen-worker interactions; indeed, the effect of multiple matings on female insect physiology has not been broadly addressed. Because it is not possible to control the natural mating behavior of queens, we used instrumental insemination and compared queens inseminated with semen from either a single drone (single-drone inseminated, or SDI) or 10 drones (multi-drone inseminated, or MDI). We used observation hives to monitor attraction of workers to SDI or MDI queens in colonies, and cage studies to monitor the attraction of workers to virgin, SDI, and MDI queen mandibular gland extracts (the main source of queen pheromone). The chemical profiles of the mandibular glands of virgin, SDI, and MDI queens were characterized using GC-MS. Finally, we measured brain expression levels in SDI and MDI queens of a gene associated with phototaxis in worker honey bees (Amfor). Here, we demonstrate for the first time that insemination quantity significantly affects mandibular gland chemical profiles, queen-worker interactions, and brain gene expression. Further research will be necessary to elucidate the mechanistic bases for these effects: insemination volume, sperm and seminal protein quantity, and genetic diversity of the sperm may all be important factors contributing to this profound change in honey bee queen physiology, queen behavior, and social interactions in the colony. PMID

  1. The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses.

    PubMed

    Uchiyama, Asako; Shimada-Beltran, Harumi; Levy, Amit; Zheng, Judy Y; Javia, Parth A; Lazarowitz, Sondra G

    2014-01-01

    Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV) and the Tobamovirus Tobacco mosaic virus (TMV) through plasmodesmata (Lewis and Lazarowitz, 2010). To determine whether SYTA has a central role in regulating the cell-to-cell trafficking of a wider range of diverse plant viruses, we extended our studies here to examine the role of SYTA in the cell-to-cell movement of additional plant viruses that employ different modes of movement, namely the Potyvirus Turnip mosaic virus (TuMV), the Caulimovirus Cauliflower mosaic virus (CaMV) and the Tobamovirus Turnip vein clearing virus (TVCV), which in contrast to TMV does efficiently infect Arabidopsis. We found that both TuMV and TVCV systemic infection, and the cell-to-cell trafficking of the their movement proteins, were delayed in the Arabidopsis Col-0 syta-1 knockdown mutant. In contrast, CaMV systemic infection was not inhibited in syta-1. Our studies show that SYTA is a key regulator of plant virus intercellular movement, being necessary for the ability of diverse cell-to-cell movement proteins encoded by Begomoviruses (CaLCuV MP), Tobamoviruses (TVCV and TMV 30K protein) and Potyviruses (TuMV P3N-PIPO) to alter PD and thereby mediate virus cell-to-cell spread. PMID:25414709

  2. The Arabidopsis synaptotagmin SYTA regulates the cell-to-cell movement of diverse plant viruses

    PubMed Central

    Uchiyama, Asako; Shimada-Beltran, Harumi; Levy, Amit; Zheng, Judy Y.; Javia, Parth A.; Lazarowitz, Sondra G.

    2014-01-01

    Synaptotagmins are a large gene family in animals that have been extensively characterized due to their role as calcium sensors to regulate synaptic vesicle exocytosis and endocytosis in neurons, and dense core vesicle exocytosis for hormone secretion from neuroendocrine cells. Thought to be exclusive to animals, synaptotagmins have recently been characterized in Arabidopsis thaliana, in which they comprise a five gene family. Using infectivity and leaf-based functional assays, we have shown that Arabidopsis SYTA regulates endocytosis and marks an endosomal vesicle recycling pathway to regulate movement protein-mediated trafficking of the Begomovirus Cabbage leaf curl virus (CaLCuV) and the Tobamovirus Tobacco mosaic virus (TMV) through plasmodesmata (Lewis and Lazarowitz, 2010). To determine whether SYTA has a central role in regulating the cell-to-cell trafficking of a wider range of diverse plant viruses, we extended our studies here to examine the role of SYTA in the cell-to-cell movement of additional plant viruses that employ different modes of movement, namely the Potyvirus Turnip mosaic virus (TuMV), the Caulimovirus Cauliflower mosaic virus (CaMV) and the Tobamovirus Turnip vein clearing virus (TVCV), which in contrast to TMV does efficiently infect Arabidopsis. We found that both TuMV and TVCV systemic infection, and the cell-to-cell trafficking of the their movement proteins, were delayed in the Arabidopsis Col-0 syta-1 knockdown mutant. In contrast, CaMV systemic infection was not inhibited in syta-1. Our studies show that SYTA is a key regulator of plant virus intercellular movement, being necessary for the ability of diverse cell-to-cell movement proteins encoded by Begomoviruses (CaLCuV MP), Tobamoviruses (TVCV and TMV 30K protein) and Potyviruses (TuMV P3N-PIPO) to alter PD and thereby mediate virus cell-to-cell spread. PMID:25414709

  3. Evolution of specialized spermatheca morphology in ant queens: insight from comparative developmental biology between ants and polistine wasps.

    PubMed

    Gotoh, Ayako; Billen, Johan; Hashim, Rosli; Ito, Fuminori

    2009-11-01

    In many ant species, the queens can keep spermatozoa alive in their spermatheca for several years, which goes along with unique morphological characteristics of the queen's spermatheca. The relative spermatheca size in ant queens is prominently larger than that in social wasps. Furthermore, the epithelium lining the spermatheca reservoir of ants consists of columnar cells in the hilar region and squamous cells in the distal region, whereas it is formed by columnar cells only in social wasps. To study the evolution of the unique spermatheca morphology in ant queens, we compared the various processes during spermatheca development between two ponerine ant species of the genus Pachycondyla (=Brachyponera) and three polistine wasp species of the genus Polistes. From histological observations, we can define four developmental events in the ant queens: (1) invagination of the spermatheca primordium, (2) the reservoir wall thickness becomes unequal, (3) the reservoir diameter doubles as the lining epithelial cells become flattened except for the hilar region, and (4) the increase in thickness of the reservoir epithelium is limited to the hilar region which doubles in thickness. In polistine wasps, the second and the third developmental events are absent and the entire epithelium of the spermatheca wall becomes thick in the final step. We therefore conclude that for ant queens the second and third steps are crucial for the enlargement of the spermatheca size, and that the second to the fourth steps are crucial for the specialization of the reservoir wall structure. PMID:19720157

  4. Inducible human immunodeficiency virus type 1 packaging cell lines.

    PubMed Central

    Yu, H; Rabson, A B; Kaul, M; Ron, Y; Dougherty, J P

    1996-01-01

    Packaging cell lines are important tools for transferring genes into eukaryotic cells. Human immunodeficiency virus type 1 (HIV-1)-based packaging cell lines are difficult to obtain, in part owing to the problem that some HIV-1 proteins are cytotoxic in a variety of cells. To overcome this, we have developed an HIV-1-based packaging cell line which has an inducible expression system. The tetracycline-inducible expression system was utilized to control the expression of the Rev regulatory protein, which in turn controls the expression of the late proteins including Gag, Pol, and Env. Western blotting (immunoblotting) demonstrated that the expression of p24gag and gp120env from the packaging cells peaked on days 6 and 7 postinduction. Reverse transcriptase activity could be detected by day 4 after induction and also peaked on days 6 and 7. Defective vector virus could be propagated, yielding titers as high as 7 x 10(3) CFU/ml, while replication-competent virus was not detectable at any time. Thus, the cell line should enable the transfer of specific genes into CD4+ cells and should be a useful tool for studying the biology of HIV-1. We have also established an inducible HIV-1 Env-expressing cell line which could be used to propagate HIV-1 vectors that require only Env in trans. The env-minus vector virus titer produced from the Env-expressing cells reached 2 x 10(4) CFU/ml. The inducible HIV-1 Env-expressing cell line should be a useful tool for the study of HIV-1 Env as well. PMID:8676479

  5. Free-virus and cell-to-cell transmission in models of equine infectious anemia virus infection.

    PubMed

    Allen, Linda J S; Schwartz, Elissa J

    2015-12-01

    Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family that infects horses and ponies. Two strains, referred to as the sensitive strain and the resistant strain, have been isolated from an experimentally-infected pony. The sensitive strain is vulnerable to neutralization by antibodies whereas the resistant strain is neutralization-insensitive. The sensitive strain mutates to the resistant strain. EIAV may infect healthy target cells via free virus or alternatively, directly from an infected target cell through cell-to-cell transfer. The proportion of transmission from free-virus or from cell-to-cell transmission is unknown. A system of ordinary differential equations (ODEs) is formulated for the virus-cell dynamics of EIAV. In addition, a Markov chain model and a branching process approximation near the infection-free equilibrium (IFE) are formulated. The basic reproduction number R0 is defined as the maximum of two reproduction numbers, R0s and R0r, one for the sensitive strain and one for the resistant strain. The IFE is shown to be globally asymptotically stable for the ODE model in a special case when the basic reproduction number is less than one. In addition, two endemic equilibria exist, a coexistence equilibrium and a resistant strain equilibrium. It is shown that if R0>1, the infection persists with at least one of the two strains. However, for small infectious doses, the sensitive strain and the resistant strain may not persist in the Markov chain model. Parameter values applicable to EIAV are used to illustrate the dynamics of the ODE and the Markov chain models. The examples highlight the importance of the proportion of cell-to-cell versus free-virus transmission that either leads to infection clearance or to infection persistence with either coexistence of both strains or to dominance by the resistant strain. PMID:25865935

  6. Human T cell lymphotropic virus-associated leukemia/lymphoma

    PubMed Central

    Ratner, Lee

    2009-01-01

    Purpose of review This article summarizes the current pathophysiologic basis for human T cell lymphotropic virus-associated leukemia/lymphoma as well as past, present, and future therapeutic options. Recent findings New studies have been published on allogeneic stem cell transplantation, arsenic trioxide, and bortezomib for this condition. Summary Studies of the molecular biology of human T cell lymphotropic virus-1-induced T cell leukemia/lymphoma have defined a critical role for oncoprotein, Tax, and activation of nuclear factor κB transcription pathways, which have provided rational approaches to improved therapy for T cell leukemia/lymphoma as well as a model for other hematopoietic malignancies characterized by nuclear factor κB activation. PMID:16093798

  7. [Dynamics of the cell cycle in human endothelial cell culture infected with influenza virus].

    PubMed

    Prochukhanova, A R; Lyublinskaya, O G; Azarenok, A A; Nazarova, A V; Zenin, V V; Zhilinskaya, I N

    2015-01-01

    Cell cycle in a culture of endothelial cells EAhy 926 infected with influenza virus was investigated. Cytometric analysis of culture, synchronized using contact inhibition, has shown that the exposure to the influenza virus in cells EAhy 926 lengthened S-phase of the cell cycle. This result has been tested and proven on culture EAhy 926 treated with nocodazole. Compared with lung carcinoma cells A549, in which influenza virus provokes the arrest of G0/G1 phase of the cycle, elongation of S-phase of cycle at a similar infection of endothelial culture EAhy 926 indicates that the influenza virus differently affects the dynamics of the cell cycle according to the origin of the infected culture. PMID:26021172

  8. Ebola virus. Two-pore channels control Ebola virus host cell entry and are drug targets for disease treatment.

    PubMed

    Sakurai, Yasuteru; Kolokoltsov, Andrey A; Chen, Cheng-Chang; Tidwell, Michael W; Bauta, William E; Klugbauer, Norbert; Grimm, Christian; Wahl-Schott, Christian; Biel, Martin; Davey, Robert A

    2015-02-27

    Ebola virus causes sporadic outbreaks of lethal hemorrhagic fever in humans, but there is no currently approved therapy. Cells take up Ebola virus by macropinocytosis, followed by trafficking through endosomal vesicles. However, few factors controlling endosomal virus movement are known. Here we find that Ebola virus entry into host cells requires the endosomal calcium channels called two-pore channels (TPCs). Disrupting TPC function by gene knockout, small interfering RNAs, or small-molecule inhibitors halted virus trafficking and prevented infection. Tetrandrine, the most potent small molecule that we tested, inhibited infection of human macrophages, the primary target of Ebola virus in vivo, and also showed therapeutic efficacy in mice. Therefore, TPC proteins play a key role in Ebola virus infection and may be effective targets for antiviral therapy. PMID:25722412

  9. Herpes simplex viruses lacking glycoprotein D are unable to inhibit virus penetration: quantitative evidence for virus-specific cell surface receptors

    SciTech Connect

    Johnson, D.C.; Ligas, M.W.

    1988-12-01

    Herpes simplex virus (HSV) glycoprotein D (gD) plays an essential role in the entry of virus into cells. HSV mutants unable to express gD were constructed. The mutants can be propagated on VD60 cells, which supply the viruses with gD; however, virus particles lacking gD were produced in mutant-infected Vero cells. Virus particles with or without gD adsorbed to a large number of sites on the cell surface; however, virions lacking gD did not enter cells. Cells pretreated with UV-inactivated virions containing gD were resistant to infection with HSV type 1 (HSV-1) and HSV-2. In contrast, cell pretreated with UV-inactivated virions lacking gD could be infected with HSV-1 and HSV-2. If infectious HSV-1 was added prior to UV-inactivated virus particles containing gD, the infectious virus entered cells and replicated. Therefore, virus particles containing gD appear to block specific cell surface receptors which are very limited in number. Particles lacking gD are presumably unable to interact with these receptors, suggesting that gD is an essential receptor-binding polypeptide.

  10. Antibodies to CD9, a Tetraspan Transmembrane Protein, Inhibit Canine Distemper Virus-Induced Cell-Cell Fusion but Not Virus-Cell Fusion

    PubMed Central

    Schmid, Erik; Zurbriggen, Andreas; Gassen, Uta; Rima, Bert; ter Meulen, Volker; Schneider-Schaulies, Jürgen

    2000-01-01

    Canine distemper virus (CDV) causes a life-threatening disease in several carnivores including domestic dogs. Recently, we identified a molecule, CD9, a member of the tetraspan transmembrane protein family, which facilitates, and antibodies to which inhibit, the infection of tissue culture cells with CDV (strain Onderstepoort). Here we describe that an anti-CD9 monoclonal antibody (MAb K41) did not interfere with binding of CDV to cells and uptake of virus. In addition, in single-step growth experiments, MAb K41 did not induce differences in the levels of viral mRNA and proteins. However, the virus release of syncytium-forming strains of CDV, the virus-induced cell-cell fusion in lytically infected cultures, and the cell-cell fusion of uninfected with persistently CDV-infected HeLa cells were strongly inhibited by MAb K41. These data indicate that anti-CD9 antibodies selectively block virus-induced cell-cell fusion, whereas virus-cell fusion is not affected. PMID:10906209

  11. Vaccinia Virus Induces Programmed Necrosis in Ovarian Cancer Cells

    PubMed Central

    Whilding, Lynsey M; Archibald, Kyra M; Kulbe, Hagen; Balkwill, Frances R; Öberg, Daniel; McNeish, Iain A

    2013-01-01

    The mechanisms by which oncolytic vaccinia virus induces tumor cell death are poorly understood. We have evaluated cell death pathways following infection of ovarian cancer cells with both wild-type and thymidine kinase-deleted (dTK) Lister strain vaccinia. We show that death does not rely upon classical apoptosis despite the appearances of some limited apoptotic features, including phosphatidylserine externalization and appearance of sub-G1 DNA populations. Vaccinia infection induces marked lipidation of LC3 proteins, but there is no general activation of the autophagic process and cell death does not rely upon autophagy induction. We show that vaccinia induces necrotic morphology on transmission electron microscopy, accompanied by marked by reductions in intracellular adenosine triphosphate, altered mitochondrial metabolism, and release of high mobility group box 1 (HMGB1) protein. This necrotic cell death appears regulated, as infection induces formation of a receptor interacting protein (RIP1)/caspase-8 complex. In addition, pharmacological inhibition of both RIP1 and substrates downstream of RIP1, including MLKL, significantly attenuate cell death. Blockade of TNF-α, however, does not alter virus efficacy, suggesting that necrosis does not result from autocrine cytokine release. Overall, these results show that, in ovarian cancer cells, vaccinia virus causes necrotic cell death that is mediated through a programmed series of events. PMID:23985697

  12. Innate Sensing of Foamy Viruses by Human Hematopoietic Cells

    PubMed Central

    Rua, Réjane; Lepelley, Alice; Gessain, Antoine

    2012-01-01

    Foamy viruses (FV) are nonpathogenic retroviruses that have cospeciated with primates for millions of years. FV can be transmitted through severe bites from monkeys to humans. Viral loads remain generally low in infected humans, and no secondary transmission has been reported. Very little is known about the ability of FV to trigger an innate immune response in human cells. A few previous reports suggested that FV do not induce type I interferon (IFN) in nonhematopoietic cells. Here, we examined how human hematopoietic cells sense FV particles and FV-infected cells. We show that peripheral blood mononuclear cells (PBMCs), plasmacytoid dendritic cells (pDCs), and the pDC-like cell line Gen2.2 detect FV, produce high levels of type I IFN, and express the IFN-stimulated gene MxA. Fewer than 20 FV-infected cells are sufficient to trigger an IFN response. Both prototypic and primary viruses stimulated IFN release. Donor cells expressing a replication-defective virus, carrying a mutated reverse transcriptase, induced IFN production by target cells as potently as wild-type virus. In contrast, an FV strain with env deleted, which does not produce viral particles, was inactive. IFN production was blocked by an inhibitor of endosomal acidification (bafilomycin A1) and by an endosomal Toll-like receptor (TLR) antagonist (A151). Silencing experiments in Gen2.2 further demonstrated that TLR7 is involved in FV recognition. Therefore, FV are potent inducers of type I IFN by pDCs and by PBMCs. This previously underestimated activation of the innate immune response may be involved in the control of viral replication in humans. PMID:22090096

  13. Preferential targeting of vesicular stomatitis virus to breast cancer cells

    SciTech Connect

    Bergman, Ira . E-mail: ira.bergman@chp.edu; Whitaker-Dowling, Patricia; Gao Yanhua; Griffin, Judith A.

    2004-12-05

    Vesicular stomatitis virus (VSV) is a candidate for development for cancer therapy. We created a recombinant replicating VSV (rrVSV) with an altered surface protein that targeted preferentially to breast cancer cells. The rrVSV genome contained a single glycoprotein (gp) gene derived from Sindbis virus. This gene expressed a chimeric Sindbis E2 binding gp and the native Sindbis E1 fusion gp. The chimeric E2 binding gp, called Sindbis-SCA-erbb2, was modified to reduce its native binding function and to contain a single chain antibody (SCA) with specificity for the human epidermal growth factor receptor Her2/neu protein, erbb2. These viruses selectively infected, replicated in and killed cells expressing erbb2. The titer of rrVSV on SKBR3 cells, a human breast cancer cell line which highly expresses erbb2 was 3.1 x 10{sup 7}/ml compared with a titer of 7.3 x 10{sup 5}/ml on 143 cells, a human osteosarcoma cell line which does not express erbb2. The titer of rrVSV on D2F2/E2 cells, a mouse mammary cancer cell line stably transfected to express human erbb2 was 2.46 x 10{sup 6}/ml compared with a titer of 5 x 10{sup 4}/ml on the parent D2F2 cells which do not express erbb2. When titered on erbb2-negative cells, non-replicating pseudotype VSV coated with Sindbis-SCA-erbb2 had <3% the titer of pseudotype VSV coated with wild type Sindbis gp indicating that the chimeric Sindbis gp had severely impaired binding to the natural receptor. Analysis of the protein composition of the rrVSV found low expression of the modified Sindbis gp on the virus.

  14. Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana

    PubMed Central

    Holmes, M J; Tan, K; Wang, Z; Oldroyd, B P; Beekman, M

    2015-01-01

    Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species. PMID:25052414

  15. Genetic reincarnation of workers as queens in the Eastern honeybee Apis cerana.

    PubMed

    Holmes, M J; Tan, K; Wang, Z; Oldroyd, B P; Beekman, M

    2015-01-01

    Thelytokous parthenogenesis, or the asexual production of female offspring, is rare in the animal kingdom, but relatively common in social Hymenoptera. However, in honeybees, it is only known to be ubiquitous in one subspecies of Apis mellifera, the Cape honeybee, A. mellifera capensis. Here we report the appearance of queen cells in two colonies of the Eastern honeybee Apis cerana that no longer contained a queen or queen-produced brood to rear queens from. A combination of microsatellite genotyping and the timing of the appearance of these individuals excluded the possibility that they had been laid by the original queen. Based on the genotypes of these individuals, thelytokous production by natal workers is the most parsimonious explanation for their existence. Thus, we present the first example of thelytoky in a honeybee outside A. mellifera. We discuss the evolutionary and ecological consequences of thelytoky in A. cerana, in particular the role thelytoky may play in the recent invasions by populations of this species. PMID:25052414

  16. Behavioral plasticity in ant queens: environmental manipulation induces aggression among normally peaceful queens in the socially polymorphic ant Leptothorax acervorum.

    PubMed

    Trettin, Jürgen; Seyferth, Thomas; Heinze, Jürgen

    2014-01-01

    The behavioral traits that shape the structure of animal societies vary considerably among species but appear to be less flexible within species or at least within populations. Populations of the ant Leptothorax acervorum differ in how queens interact with other queens. Nestmate queens from extended, homogeneous habitats tolerate each other and contribute quite equally to the offspring of the colony (polygyny: low reproductive skew). In contrast, nestmate queens from patchy habitats establish social hierarchies by biting and antennal boxing, and eventually only the top-ranking queen of the colony lays eggs (functional monogyny: high reproductive skew). Here we investigate whether queen-queen behavior is fixed within populations or whether aggression and high skew can be elicited by manipulation of socio-environmental factors in colonies from low skew populations. An increase of queen/worker ratio and to a lesser extent food limitation elicited queen-queen antagonism in polygynous colonies from Nürnberger Reichswald similar to that underlying social and reproductive hierarchies in high-skew populations from Spain, Japan, and Alaska. In manipulated colonies, queens differed more in ovarian status than in control colonies. This indicates that queens are in principle capable of adapting the magnitude of reproductive skew to environmental changes in behavioral rather than evolutionary time. PMID:24743352

  17. Identification of Cell Surface Molecules Involved in Dystroglycan-Independent Lassa Virus Cell Entry

    PubMed Central

    Ströher, Ute; Ebihara, Hideki; Feldmann, Heinz

    2012-01-01

    Although O-mannosylated dystroglycan is a receptor for Lassa virus, a causative agent of Lassa fever, recent findings suggest the existence of an alternative receptor(s). Here we identified four molecules as receptors for Lassa virus: Axl and Tyro3, from the TAM family, and dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) and liver and lymph node sinusoidal endothelial calcium-dependent lectin (LSECtin), from the C-type lectin family. These molecules enhanced the binding of Lassa virus to cells and mediated infection independently of dystroglycan. Axl- or Tyro3-mediated infection required intracellular signaling via the tyrosine kinase activity of Axl or Tyro3, whereas DC-SIGN- or LSECtin-mediated infection and binding were dependent on a specific carbohydrate and on ions. The identification of these four molecules as Lassa virus receptors advances our understanding of Lassa virus cell entry. PMID:22156524

  18. Herpes B Virus Utilizes Human Nectin-1 but Not HVEM or PILRα for Cell-Cell Fusion and Virus Entry

    PubMed Central

    Fan, Qing; Amen, Melanie; Harden, Mallory; Severini, Alberto; Griffiths, Anthony

    2012-01-01

    To investigate the requirements of herpesvirus entry and fusion, the four homologous glycoproteins necessary for herpes simplex virus (HSV) fusion were cloned from herpes B virus (BV) (or macacine herpesvirus 1, previously known as cercopithecine herpesvirus 1) and cercopithecine herpesvirus 2 (CeHV-2), both related simian simplexviruses belonging to the alphaherpesvirus subfamily. Western blots and cell-based enzyme-linked immunosorbent assay (ELISA) showed that glycoproteins gB, gD, and gH/gL were expressed in whole-cell lysates and on the cell surface. Cell-cell fusion assays indicated that nectin-1, an HSV-1 gD receptor, mediated fusion of cells expressing glycoproteins from both BV and CeHV-2. However, herpesvirus entry mediator (HVEM), another HSV-1 gD receptor, did not facilitate BV- and CeHV-2-induced cell-cell fusion. Paired immunoglobulin-like type 2 receptor alpha (PILRα), an HSV-1 gB fusion receptor, did not mediate fusion of cells expressing glycoproteins from either simian virus. Productive infection with BV was possible only with nectin-1-expressing cells, indicating that nectin-1 mediated entry while HVEM and PILRα did not function as entry receptors. These results indicate that these alphaherpesviruses have differing preferences for entry receptors. The usage of the HSV-1 gD receptor nectin-1 may explain interspecies transfer of the viruses, and altered receptor usage may result in altered virulence, tropism, or pathogenesis in the new host. A heterotypic cell fusion assay resulting in productive fusion may provide insight into interactions that occur to trigger fusion. These findings may be of therapeutic significance for control of deadly BV infections. PMID:22345445

  19. Women in History--Queen Liliuokalani

    ERIC Educational Resources Information Center

    Koeppe, Tina

    2007-01-01

    This article profiles Queen Liliuokalani, Hawaii's last monarch. Liliuokalani was born in Hawaii in 1838 into the family of a high chief. She attended the Royal School, run by American missionaries, and received a high quality education and learned to love music, writing and politics. Liliuokalani was given the Christian name "Lydia" as a child.…

  20. Queen Margaret University College's Sustainable, Community Campus

    ERIC Educational Resources Information Center

    Woodman, Susan

    2006-01-01

    The new campus of Queen Margaret University College in the United Kingdom is designed to be a sustainable educational and community resource. Early consultation with students and staff on the campus design revealed a strong desire for a sustainable environment, with plenty of green space for all to enjoy. In response to this, the design focuses on…

  1. INFECTIVITY AND PERSISTENCE OF VESICULAR STOMATITIS VIRUS IN CULICOIDES CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biting midge, Culicoides sonorensis, was recently shown to be a biologically competent vector for the arbovirus, vesicular stomatitis virus (VSV). While arboviruses can be extremely pathogenic to mammalian cells, they typically do not exert deleterious effects on their insect vectors. Infectio...

  2. Senescence affects endothelial cells susceptibility to dengue virus infection.

    PubMed

    AbuBakar, Sazaly; Shu, Meng-Hooi; Johari, Jefree; Wong, Pooi-Fong

    2014-01-01

    Alteration in the endothelium leading to increased vascular permeability contributes to plasma leakage seen in dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). An earlier study showed that senescent endothelial cells (ECs) altered the ECs permeability. Here we investigated the susceptibility of senescing human umbilical vein endothelial cells (HUVECs) to dengue virus infection and determined if dengue virus infection induces HUVECs senescence. Our results suggest that DENV type-2 (DENV-2) foci forming unit (FFU) and extracellular virus RNA copy number were reduced by at least 35% and 85% in infection of the intermediate young and early senescent HUVECs, respectively, in comparison to infection of young HUVECs. No to low infectivity was recovered from infection of late senescent HUVECs. DENV infection also increases the percentage of HUVECs expressing senescence-associated (SA)-β-gal, cells arrested at the G2/M phase or 4N DNA content stage and cells with enlarged morphology, indicative of senescing cells. Alteration of HUVECs morphology was recorded using impedance-based real-time cell analysis system following DENV-2 infection. These results suggest that senescing HUVECs do not support DENV infection and DENV infection induces HUVECs senescence. The finding highlights the possible role of induction of senescence in DENV infection of the endothelial cells. PMID:24782642

  3. Replication of the Moloney murine sarcoma-leukemia virus in XC cells.

    PubMed

    Trowbridge, S T; Benyesh-Melnick, M; Biswal, N

    1973-01-01

    The XC rat cell line was found to support the replication of a strain of the Moloney murine sarcoma-leukemia virus. In growth curve experiments cytopathology was paralleled by the production of murine sarcoma virus and leukemia virus progeny having the biologic, antigenic, and biophysical properties of the infecting virus. PMID:4346280

  4. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    PubMed Central

    Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments. PMID:19096510

  5. Isolation of a new herpes virus from human CD4 sup + T cells

    SciTech Connect

    Frenkel, N.; Schirmer, E.C.; Wyatt, L.S.; Katsafanas, G.; Roffman, E.; Danovich, R.M. ); June, C.H. )

    1990-01-01

    A new human herpes virus has been isolated from CD4{sup +} T cells purified from peripheral blood mononuclear cells of a healthy individual (RK), following incubation of the cells under conditions promoting T-cell activation. The virus could not be recovered from nonactivated cells. Cultures of lymphocytes infected with the RK virus exhibited a cytopathic effect, and electron microscopic analyses revealed a characteristic herpes virus structure. RK virus DNA did not hybridize with large probes derived from herpes simplex virus, Epstein-Barr virus, varicella-zoster virus, and human cytomegalovirus. The genetic relatedness of the RK virus to the recently identified T-lymphotropic human herpes virus 6 (HHV-6) was investigated by restriction enzyme analyses using 21 different enzymes and by blot hydridization analyses using 11 probes derived from two strains of HHV-6 (Z29 and U1102). Whereas the two HHV-6 strains exhibited only limited restriction enzyme polymorphism, cleavage of the RK virus DNA yielded distinct patterns. Of the 11 HHV-6 DNA probes tested, only 6 cross-hybridized with DNA fragments derived from the RK virus. Taken together, the maximal homology amounted to 31 kilobases of the 75 kilobases tested. The authors conclude that the RK virus is distinct from previously characterized human herpesviruses. The authors propose to designate it as the prototype of a new herpes virus, the seventh human herpes virus identified to date.

  6. Entry of Vesicular Stomatitis Virus into L Cells

    PubMed Central

    Heine, Jochen W.; Schnaitman, Carl A.

    1971-01-01

    Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of 3H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm. Images PMID:4332145

  7. Surface lipids of queen-laid eggs do not regulate queen production in a fission-performing ant

    NASA Astrophysics Data System (ADS)

    Ruel, Camille; Lenoir, Alain; Cerdá, Xim; Boulay, Raphaël

    2013-01-01

    In animal societies, most collective and individual decision making depends on the presence of reproductive individuals. The efficient transmission of information among reproductive and non-reproductive individuals is therefore a determinant of colony organization. In social insects, the presence of a queen modulates multiple colonial activities. In many species, it negatively affects worker reproduction and the development of diploid larvae into future queens. The queen mostly signals her presence through pheromone emission, but the means by which these chemicals are distributed in the colony are still unclear. In several ant species, queen-laid eggs are the vehicle of the queen signal. The aim of this study was to investigate whether queen-laid eggs of the ant Aphaenogaster senilis possess queen-specific cuticular hydrocarbons and/or Dufour or poison gland compounds, and whether the presence of eggs inhibited larval development into queens. Our results show that the queen- and worker-laid eggs shared cuticular and Dufour hydrocarbons with the adults; however, their poison gland compounds were not similar. Queen-laid eggs had more dimethylalkanes and possessed a queen-specific mixture of cuticular hydrocarbons composed of 3,11 + 3,9 + 3,7-dimethylnonacosane, in higher proportions than did worker-laid eggs. Even though the queen-laid eggs were biochemically similar to the queen, their addition to experimentally queenless groups did not prevent the development of new queens. More studies are needed on the means by which queen ant pheromones are transmitted in the colony, and how these mechanisms correlates with life history traits.

  8. Surface lipids of queen-laid eggs do not regulate queen production in a fission-performing ant.

    PubMed

    Ruel, Camille; Lenoir, Alain; Cerdá, Xim; Boulay, Raphaël

    2013-01-01

    In animal societies, most collective and individual decision making depends on the presence of reproductive individuals. The efficient transmission of information among reproductive and non-reproductive individuals is therefore a determinant of colony organization. In social insects, the presence of a queen modulates multiple colonial activities. In many species, it negatively affects worker reproduction and the development of diploid larvae into future queens. The queen mostly signals her presence through pheromone emission, but the means by which these chemicals are distributed in the colony are still unclear. In several ant species, queen-laid eggs are the vehicle of the queen signal. The aim of this study was to investigate whether queen-laid eggs of the ant Aphaenogaster senilis possess queen-specific cuticular hydrocarbons and/or Dufour or poison gland compounds, and whether the presence of eggs inhibited larval development into queens. Our results show that the queen- and worker-laid eggs shared cuticular and Dufour hydrocarbons with the adults; however, their poison gland compounds were not similar. Queen-laid eggs had more dimethylalkanes and possessed a queen-specific mixture of cuticular hydrocarbons composed of 3,11 + 3,9 + 3,7-dimethylnonacosane, in higher proportions than did worker-laid eggs. Even though the queen-laid eggs were biochemically similar to the queen, their addition to experimentally queenless groups did not prevent the development of new queens. More studies are needed on the means by which queen ant pheromones are transmitted in the colony, and how these mechanisms correlates with life history traits. PMID:23224071

  9. Queen pheromone regulates egg production in a termite.

    PubMed

    Yamamoto, Yuuka; Matsuura, Kenji

    2011-10-23

    In social insects, resource allocation is a key factor that influences colony survival and growth. Optimal allocation to queens and brood is essential for maximum colony productivity, requiring colony members to have information on the total reproductive power in colonies. However, the mechanisms regulating egg production relative to the current labour force for brood care remain poorly known. Recently, a volatile chemical was identified as a termite queen pheromone that inhibits the differentiation of new neotenic reproductives (secondary reproductives developed from nymphs or workers) in Reticulitermes speratus. The same volatile chemical is also emitted by eggs. This queen pheromone would therefore be expected to act as an honest message of the reproductive power about queens. In this study, we examined how the queen pheromone influences the reproductive rate of queens in R. speratus. We compared the number of eggs produced by each queen between groups with and without exposure to artificial queen pheromone. Exposure to the pheromone resulted in a significant decrease in egg production in both single-queen and multiple-queen groups. This is the first report supporting the role of queen pheromones as a signal regulating colony-level egg production, using synthetically derived compounds in a termite. PMID:21543395

  10. Assessing the mating 'health' of commercial honey bee queens.

    PubMed

    Tarpy, David R; Keller, Jennifer J; Caren, Joel R; Delaney, Deborah A

    2012-02-01

    Honey bee queens mate with multiple males, which increases the total genetic diversity within colonies and has been shown to confer numerous benefits for colony health and productivity. Recent surveys of beekeepers have suggested that 'poor queens' are a top management concern, thus investigating the reproductive quality and mating success of commercially produced honey bee queens is warranted. We purchased 80 commercially produced queens from large queen breeders in California and measured them for their physical size (fresh weigh and thorax width), insemination success (stored sperm counts and sperm viability), and mating number (determined by patriline genotyping of worker offspring). We found that queens had an average of 4.37 +/- 1.446 million stored sperm in their spermathecae with an average viability of 83.7 +/- 13.33%. We also found that the tested queens had mated with a high number of drones (average effective paternity frequency: 17.0 +/- 8.98). Queen "quality" significantly varied among commercial sources for physical characters but not for mating characters. These findings suggest that it may be more effective to improve overall queen reproductive potential by culling lower-quality queens rather than systematically altering current queen production practices. PMID:22420250

  11. Oxidative stress modulation in hepatitis C virus infected cells

    PubMed Central

    Lozano-Sepulveda, Sonia A; Bryan-Marrugo, Owen L; Cordova-Fletes, Carlos; Gutierrez-Ruiz, Maria C; Rivas-Estilla, Ana M

    2015-01-01

    Hepatitis C virus (HCV) replication is associated with the endoplasmic reticulum, where the virus can induce cellular stress. Oxidative cell damage plays an important role in HCV physiopathology. Oxidative stress is triggered when the concentration of oxygen species in the extracellular or intracellular environment exceeds antioxidant defenses. Cells are protected and modulate oxidative stress through the interplay of intracellular antioxidant agents, mainly glutathione system (GSH) and thioredoxin; and antioxidant enzyme systems such as superoxide dismutase, catalase, GSH peroxidase, and heme oxygenase-1. Also, the use of natural and synthetic antioxidants (vitamin C and E, N-acetylcysteine, glycyrrhizin, polyenylphosphatidyl choline, mitoquinone, quercetin, S-adenosylmethionine and silymarin) has already shown promising results as co-adjuvants in HCV therapy. Despite all the available information, it is not known how different agents with antiviral activity can interfere with the modulation of the cell redox state induced by HCV and decrease viral replication. This review describes an evidence-based consensus on molecular mechanisms involved in HCV replication and their relationship with cell damage induced by oxidative stress generated by the virus itself and cell antiviral machinery. It also describes some molecules that modify the levels of oxidative stress in HCV-infected cells. PMID:26692473

  12. Measles Virus Matrix Protein Inhibits Host Cell Transcription.

    PubMed

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  13. Measles Virus Matrix Protein Inhibits Host Cell Transcription

    PubMed Central

    Yu, Xuelian; Shahriari, Shadi; Li, Hong-Mei; Ghildyal, Reena

    2016-01-01

    Measles virus (MeV) is a highly contagious virus that still causes annual epidemics in developing countries despite the availability of a safe and effective vaccine. Additionally, importation from endemic countries causes frequent outbreaks in countries where it has been eliminated. The M protein of MeV plays a key role in virus assembly and cytopathogenesis; interestingly, M is localised in nucleus, cytoplasm and membranes of infected cells. We have used transient expression of M in transfected cells and in-cell transcription assays to show that only some MeV M localizes to the nucleus, in addition to cell membranes and the cytoplasm as previously described, and can inhibit cellular transcription via binding to nuclear factors. Additionally, MeV M was able to inhibit in vitro transcription in a dose-dependent manner. Importantly, a proportion of M is also localized to nucleus of MeV infected cells at early times in infection, correlating with inhibition of cellular transcription. Our data show, for the first time, that MeV M may play a role early in infection by inhibiting host cell transcription. PMID:27551716

  14. Alteration of cell cycle progression by Sindbis virus infection

    SciTech Connect

    Yi, Ruirong; Saito, Kengo; Isegawa, Naohisa; Shirasawa, Hiroshi

    2015-07-10

    We examined the impact of Sindbis virus (SINV) infection on cell cycle progression in a cancer cell line, HeLa, and a non-cancerous cell line, Vero. Cell cycle analyses showed that SINV infection is able to alter the cell cycle progression in both HeLa and Vero cells, but differently, especially during the early stage of infection. SINV infection affected the expression of several cell cycle regulators (CDK4, CDK6, cyclin E, p21, cyclin A and cyclin B) in HeLa cells and caused HeLa cells to accumulate in S phase during the early stage of infection. Monitoring SINV replication in HeLa and Vero cells expressing cell cycle indicators revealed that SINV which infected HeLa cells during G{sub 1} phase preferred to proliferate during S/G{sub 2} phase, and the average time interval for viral replication was significantly shorter in both HeLa and Vero cells infected during G{sub 1} phase than in cells infected during S/G{sub 2} phase. - Highlights: • SINV infection was able to alter the cell cycle progression of infected cancer cells. • SINV infection can affect the expression of cell cycle regulators. • SINV infection exhibited a preference for the timing of viral replication among the cell cycle phases.

  15. The Potato virus X TGBp3 protein associates with the ER network for virus cell-to-cell movement

    NASA Technical Reports Server (NTRS)

    Krishnamurthy, Konduru; Heppler, Marty; Mitra, Ruchira; Blancaflor, Elison; Payton, Mark; Nelson, Richard S.; Verchot-Lubicz, Jeanmarie

    2003-01-01

    Potato virus X (PVX) TGBp3 is required for virus cell-to-cell movement. Cell-to-cell movement of TGBp3 was studied using biolistic bombardment of plasmids expressing GFP:TGBp3. TGBp3 moves between cells in Nicotiana benthamiana, but requires TGBp1 to move in N. tabacum leaves. In tobacco leaves GFP:TGBp3 accumulated in a pattern resembling the endoplasmic reticulum (ER). To determine if the ER network is important for GFP:TGBp3 and for PVX cell-to-cell movement, a single mutation inhibiting membrane binding of TGBp3 was introduced into GFP:TGBp3 and into PVX. This mutation disrupted movement of GFP:TGBp3 and PVX. Brefeldin A, which disrupts the ER network, also inhibited GFP:TGBp3 movement in both Nicotiana species. Two deletion mutations, that do not affect membrane binding, hindered GFP:TGBp3 and PVX cell-to-cell movement. Plasmids expressing GFP:TGBp2 and GFP:TGBp3 were bombarded to several other PVX hosts and neither protein moved between adjacent cells. In most hosts, TGBp2 or TGBp3 cannot move cell-to-cell.

  16. Sperm use economy of honeybee (Apis mellifera) queens.

    PubMed

    Baer, Boris; Collins, Jason; Maalaps, Kristiina; den Boer, Susanne P A

    2016-05-01

    The queens of eusocial ants, bees, and wasps only mate during a very brief period early in life to acquire and store a lifetime supply of sperm. As sperm cannot be replenished, queens have to be highly economic when using stored sperm to fertilize eggs, especially in species with large and long-lived colonies. However, queen fertility has not been studied in detail, so that we have little understanding of how economic sperm use is in different species, and whether queens are able to influence their sperm use. This is surprising given that sperm use is a key factor of eusocial life, as it determines the fecundity and longevity of queens and therefore colony fitness. We quantified the number of sperm that honeybee (Apis mellifera) queens use to fertilize eggs. We examined sperm use in naturally mated queens of different ages and in queens artificially inseminated with different volumes of semen. We found that queens are remarkably efficient and only use a median of 2 sperm per egg fertilization, with decreasing sperm use in older queens. The number of sperm in storage was always a significant predictor for the number of sperm used per fertilization, indicating that queens use a constant ratio of spermathecal fluid relative to total spermathecal volume of 2.364 × 10(-6) to fertilize eggs. This allowed us to calculate a lifetime fecundity for honeybee queens of around 1,500,000 fertilized eggs. Our data provide the first empirical evidence that honeybee queens do not manipulate sperm use, and fertilization failures in worker-destined eggs are therefore honest signals that workers can use to time queen replacement, which is crucial for colony performance and fitness. PMID:27217944

  17. Hsp90 inhibitors reduce influenza virus replication in cell culture

    SciTech Connect

    Chase, Geoffrey; Deng, Tao; Fodor, Ervin; Leung, B.W.; Mayer, Daniel; Schwemmle, Martin Brownlee, George

    2008-08-01

    The viral RNA polymerase complex of influenza A virus consists of three subunits PB1, PB2 and PA. Recently, the cellular chaperone Hsp90 was shown to play a role in nuclear import and assembly of the trimeric polymerase complex by binding to PB1 and PB2. Here we show that Hsp90 inhibitors, geldanamycin or its derivative 17-AAG, delay the growth of influenza virus in cell culture resulting in a 1-2 log reduction in viral titre early in infection. We suggest that this is caused by the reduced half-life of PB1 and PB2 and inhibition of nuclear import of PB1 and PA which lead to reduction in viral RNP assembly. Hsp90 inhibitors may represent a new class of antiviral compounds against influenza viruses.

  18. Genome rearrangement affects RNA virus adaptability on prostate cancer cells.

    PubMed

    Pesko, Kendra; Voigt, Emily A; Swick, Adam; Morley, Valerie J; Timm, Collin; Yin, John; Turner, Paul E

    2015-01-01

    Gene order is often highly conserved within taxonomic groups, such that organisms with rearranged genomes tend to be less fit than wild type gene orders, and suggesting natural selection favors genome architectures that maximize fitness. But it is unclear whether rearranged genomes hinder adaptability: capacity to evolutionarily improve in a new environment. Negative-sense non-segmented RNA viruses (order Mononegavirales) have specific genome architecture: 3' UTR - core protein genes - envelope protein genes - RNA-dependent RNA-polymerase gene - 5' UTR. To test how genome architecture affects RNA virus evolution, we examined vesicular stomatitis virus (VSV) variants with the nucleocapsid (N) gene moved sequentially downstream in the genome. Because RNA polymerase stuttering in VSV replication causes greater mRNA production in upstream genes, N gene translocation toward the 5' end leads to stepwise decreases in N transcription, viral replication and progeny production, and also impacts the activation of type 1 interferon mediated antiviral responses. We evolved VSV gene-order variants in two prostate cancer cell lines: LNCap cells deficient in innate immune response to viral infection, and PC-3 cells that mount an IFN stimulated anti-viral response to infection. We observed that gene order affects phenotypic adaptability (reproductive growth; viral suppression of immune function), especially on PC-3 cells that strongly select against virus infection. Overall, populations derived from the least-fit ancestor (most-altered N position architecture) adapted fastest, consistent with theory predicting populations with low initial fitness should improve faster in evolutionary time. Also, we observed correlated responses to selection, where viruses improved across both hosts, rather than suffer fitness trade-offs on unselected hosts. Whole genomics revealed multiple mutations in evolved variants, some of which were conserved across selective environments for a given gene

  19. Screening of Natural Waters for Viruses Which Infect Chlorella Cells

    PubMed Central

    Yamada, Takashi; Higashiyama, Takanobu; Fukuda, Takao

    1991-01-01

    By using a plaque assay with the unicellular green alga Chlorella sp. strain NC64A as a host, viruses were screened from natural pond waters collected in Kyoto and Higashi-Hiroshima, Japan. From some samples tested, two kinds of plaques, large (φ = 6 to 10 mm) and small (φ = 2 to 3 mm), were detected with various frequencies. The frequency of plaques in each of the water sources was seasonal; generally, it reached a peak value (8,000 PFU/ml) in May and gradually decreased to the limit of detection (<1) in November before increasing again in early spring. Electron microscopy revealed that the purified and negatively stained viruses were very large (125 to 200 nm) icosahedral particles. The genome isolated from these particles was always a linear double-stranded DNA of 340 to 370 kbp. Electrophoresis patterns of the DNA fragments produced by digestion with restriction enzymes differed considerably from plaque to plaque, even for plaques from the same water source. However, Southern hybridization showed strong homology among all of the virus DNAs tested, indicating relatedness of those viruses. A possible use of the Chlorella virus assay system to monitor the natural population of algal cells and water quality is discussed. Images PMID:16348596

  20. Molecular mechanisms of Ebola virus pathogenesis: focus on cell death.

    PubMed

    Falasca, L; Agrati, C; Petrosillo, N; Di Caro, A; Capobianchi, M R; Ippolito, G; Piacentini, M

    2015-08-01

    Ebola virus (EBOV) belongs to the Filoviridae family and is responsible for a severe disease characterized by the sudden onset of fever and malaise accompanied by other non-specific signs and symptoms; in 30-50% of cases hemorrhagic symptoms are present. Multiorgan dysfunction occurs in severe forms with a mortality up to 90%. The EBOV first attacks macrophages and dendritic immune cells. The innate immune reaction is characterized by a cytokine storm, with secretion of numerous pro-inflammatory cytokines, which induces a huge number of contradictory signals and hurts the immune cells, as well as other tissues. Other highly pathogenic viruses also trigger cytokine storms, but Filoviruses are thought to be particularly lethal because they affect a wide array of tissues. In addition to the immune system, EBOV attacks the spleen and kidneys, where it kills cells that help the body to regulate its fluid and chemical balance and that make proteins that help the blood to clot. In addition, EBOV causes liver, lungs and kidneys to shut down their functions and the blood vessels to leak fluid into surrounding tissues. In this review, we analyze the molecular mechanisms at the basis of Ebola pathogenesis with a particular focus on the cell death pathways induced by the virus. We also discuss how the treatment of the infection can benefit from the recent experience of blocking/modulating cell death in human degenerative diseases. PMID:26024394

  1. Molecular mechanisms of Ebola virus pathogenesis: focus on cell death

    PubMed Central

    Falasca, L; Agrati, C; Petrosillo, N; Di Caro, A; Capobianchi, M R; Ippolito, G; Piacentini, M

    2015-01-01

    Ebola virus (EBOV) belongs to the Filoviridae family and is responsible for a severe disease characterized by the sudden onset of fever and malaise accompanied by other non-specific signs and symptoms; in 30–50% of cases hemorrhagic symptoms are present. Multiorgan dysfunction occurs in severe forms with a mortality up to 90%. The EBOV first attacks macrophages and dendritic immune cells. The innate immune reaction is characterized by a cytokine storm, with secretion of numerous pro-inflammatory cytokines, which induces a huge number of contradictory signals and hurts the immune cells, as well as other tissues. Other highly pathogenic viruses also trigger cytokine storms, but Filoviruses are thought to be particularly lethal because they affect a wide array of tissues. In addition to the immune system, EBOV attacks the spleen and kidneys, where it kills cells that help the body to regulate its fluid and chemical balance and that make proteins that help the blood to clot. In addition, EBOV causes liver, lungs and kidneys to shut down their functions and the blood vessels to leak fluid into surrounding tissues. In this review, we analyze the molecular mechanisms at the basis of Ebola pathogenesis with a particular focus on the cell death pathways induced by the virus. We also discuss how the treatment of the infection can benefit from the recent experience of blocking/modulating cell death in human degenerative diseases. PMID:26024394

  2. Hemagglutinin of influenza A virus binds specifically to cell surface nucleolin and plays a role in virus internalization.

    PubMed

    Chan, Che-Man; Chu, Hin; Zhang, Anna Jinxia; Leung, Lai-Han; Sze, Kong-Hung; Kao, Richard Yi-Tsun; Chik, Kenn Ka-Heng; To, Kelvin Kai-Wang; Chan, Jasper Fuk-Woo; Chen, Honglin; Jin, Dong-Yan; Liu, Liang; Yuen, Kwok-Yung

    2016-07-01

    The hemagglutinin (HA) protein of influenza A virus initiates cell entry by binding to sialic acids on target cells. In the current study, we demonstrated that in addition to sialic acids, influenza A/Puerto Rico/8/34 H1N1 (PR8) virus HA specifically binds to cell surface nucleolin (NCL). The interaction between HA and NCL was initially revealed with virus overlay protein binding assay (VOPBA) and subsequently verified with co-immunoprecipitation. Importantly, inhibiting cell surface NCL with NCL antibody, blocking PR8 viruses with purified NCL protein, or depleting endogenous NCL with siRNA all substantially reduced influenza virus internalization. We further demonstrated that NCL was a conserved cellular factor required for the entry of multiple influenza A viruses, including H1N1, H3N2, H5N1, and H7N9. Overall, our findings identified a novel role of NCL in influenza virus life cycle and established NCL as one of the host cell surface proteins for the entry of influenza A virus. PMID:27085069

  3. Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses.

    PubMed Central

    Bess, J W; Powell, P J; Issaq, H J; Schumack, L J; Grimes, M K; Henderson, L E; Arthur, L O

    1992-01-01

    Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses. Images PMID:1731111

  4. Modeling multiple infection of cells by viruses: challenges and insights

    PubMed Central

    Phan, Dustin; Wodarz, Dominik

    2015-01-01

    The multiple infection of cells with several copies of a given virus has been demonstrated in experimental systems, and has been subject to previous mathematical modeling approaches. Such models, especially those based on ordinary differential equations, can be characterized by difficulties and pitfalls. One such difficulty arises from what we refer to as multiple infection cascades. That is, such models subdivide the infected cell population into sub-populations that are carry i viruses, and each sub-population can in principle always be further infected to contain i+1 viruses. In order to study the model with numerical simulations, the infection cascade needs to be cut artificially, and this can influence the results. This is shown here in the context of the simplest setting that involves a single, homogeneous virus population. If the viral replication rate is sufficiently fast, then most infected cells will accumulate in the last member of the infection cascade, leading to incorrect numerical results. This can be observed even with relatively long infection cascades, and in this case computational costs associated with a sufficiently long infection cascade can render this approach impractical. We subsequently examine a more complex scenario where two virus types / strains with different fitness are allowed to compete. Again, we find that the length of the infection cascade can have a crucial influence on the results. Competitive exclusion can be observed for shorter infection cascades, while coexistence can be observed for longer infection cascades. More subtly, the length of the infection cascade can influence the equilibrium level of the populations in numerical simulations. Studying the model in a parameter regime where an increase in the infection cascade length does not influence the results, we examine the effect of multiple infection on the outcome of competition. We find that multiple infection can promote coexistence of virus types if there is a degree

  5. Passive carriage of rabies virus by dendritic cells.

    PubMed

    Senba, Kazuyo; Matsumoto, Takashi; Yamada, Kentaro; Shiota, Seiji; Iha, Hidekatsu; Date, Yukari; Ohtsubo, Motoaki; Nishizono, Akira

    2013-01-01

    The rabies virus (RABV) is highly neurotropic and it uses evasive strategies to successfully evade the host immune system. Because rabies is often fatal, understanding the basic processes of the virus-host interactions, particularly in the initial events of infection, is critical for the design of new therapeutic approaches to target RABV. Here, we examined the possible role of dendritic cells (DCs) in the transmission of RABV to neural cells at peripheral site of exposure. Viral replication only occurred at a low level in the DC cell line, JAWS II, after its infection with either pathogenic RABV (CVS strain) or low-pathogenic RABV (ERA strain), and no progeny viruses were produced in the culture supernatants. However, both viral genomic RNAs were retained in the long term after infection and maintained their infectivity. The biggest difference between CVS and ERA was in their ability to induce type I interferons. Although the ERA-infected JAWS II cells exhibited cytopathic effect and were apparently killed by normal spleen cells in vitro, the CVS-infected JAWS II cells showed milder cytopathic effect and less lysis when cocultured with spleen cells. Strongly increased expression of major histocompatibility complex classes I, costimulatory molecules (CD80 and CD86), type I interferons and Toll- like receptor 3, and was observed only in the ERA-inoculated JAWS II cells and not in those inoculated with CVS. During the silencing of the cellular immune response in the DCs, the pathogenic CVS strain cryptically maintained an infectious viral genome and was capable of transmitting infectious RABV to permissive neural cells. These findings demonstrate that DCs may play a role in the passive carriage of RABV during natural rabies infections. PMID:24024103

  6. Infection of Mosquito Cells (C6/36) by Dengue-2 Virus Interferes with Subsequent Infection by Yellow Fever Virus.

    PubMed

    Abrao, Emiliana Pereira; da Fonseca, Benedito Antônio Lopes

    2016-02-01

    Dengue is one of the most important diseases caused by arboviruses in the world. Yellow fever is another arthropod-borne disease of great importance to public health that is endemic to tropical regions of Africa and the Americas. Both yellow fever and dengue viruses are flaviviruses transmitted by Aedes aegypti mosquitoes, and then, it is reasonable to consider that in a given moment, mosquito cells could be coinfected by both viruses. Therefore, we decided to evaluate if sequential infections of dengue and yellow fever viruses (and vice-versa) in mosquito cells could affect the virus replication patterns. Using immunofluorescence and real-time PCR-based replication assays in Aedes albopictus C6/36 cells with single or sequential infections with both viruses, we demonstrated the occurrence of viral interference, also called superinfection exclusion, between these two viruses. Our results show that this interference pattern is particularly evident when cells were first infected with dengue virus and subsequently with yellow fever virus (YFV). Reduction in dengue virus replication, although to a lower extent, was also observed when C6/36 cells were initially infected with YFV followed by dengue virus infection. Although the importance that these findings have on nature is unknown, this study provides evidence, at the cellular level, of the occurrence of replication interference between dengue and yellow fever viruses and raises the question if superinfection exclusion could be a possible explanation, at least partially, for the reported lack of urban yellow fever occurrence in regions where a high level of dengue transmission occurs. PMID:26808727

  7. Colony strength and queen replacement in Melipona marginata (Apidae: Meliponini).

    PubMed

    Kleinert, A de M P

    2005-08-01

    Physogastric queens of Melipona marginata were removed from their colonies in order to verify the acceptance of a new queen by workers. Colony strength was evaluated according to queen oviposition rate and comb diameters. Replacement was observed seven times. Its occurrence and speed related positively to colony strength, independently of queen's age. In weak colonies, queen replacement was observed only once, following colony population increase that occurred after introduction of combs from another colony. Worker oviposition after queen removal was observed three times: in a strong colony with virgin queens and males, and in two of the weak colonies. In the first two or three days of new queen oviposition, during which most of the eggs were eaten by the queen, worker oviposition preceded almost all provisioning and oviposition processes (POPs). After this period, worker oviposition decreased until it reached around 25% of the POPs. Daily oviposition rate of young queens decreased or was even interrupted by hatching of their first brood. PMID:16341425

  8. Remote Activation of Host Cell DNA Synthesis in Uninfected Cells Signaled by Infected Cells in Advance of Virus Transmission

    PubMed Central

    Schmidt, Nora; Hennig, Thomas; Serwa, Remigiusz A.; Marchetti, Magda

    2015-01-01

    ABSTRACT Viruses modulate cellular processes and metabolism in diverse ways, but these are almost universally studied in the infected cell itself. Here, we study spatial organization of DNA synthesis during multiround transmission of herpes simplex virus (HSV) using pulse-labeling with ethynyl nucleotides and cycloaddition of azide fluorophores. We report a hitherto unknown and unexpected outcome of virus-host interaction. Consistent with the current understanding of the single-step growth cycle, HSV suppresses host DNA synthesis and promotes viral DNA synthesis in spatially segregated compartments within the cell. In striking contrast, during progressive rounds of infection initiated at a single cell, we observe that infection induces a clear and pronounced stimulation of cellular DNA replication in remote uninfected cells. This induced DNA synthesis was observed in hundreds of uninfected cells at the extended border, outside the perimeter of the progressing infection. Moreover, using pulse-chase analysis, we show that this activation is maintained, resulting in a propagating wave of host DNA synthesis continually in advance of infection. As the virus reaches and infects these activated cells, host DNA synthesis is then shut off and replaced with virus DNA synthesis. Using nonpropagating viruses or conditioned medium, we demonstrate a paracrine effector of uninfected cell DNA synthesis in remote cells continually in advance of infection. These findings have significant implications, likely with broad applicability, for our understanding of the ways in which virus infection manipulates cell processes not only in the infected cell itself but also now in remote uninfected cells, as well as of mechanisms governing host DNA synthesis. IMPORTANCE We show that during infection initiated by a single particle with progressive cell-cell virus transmission (i.e., the normal situation), HSV induces host DNA synthesis in uninfected cells, mediated by a virus-induced paracrine

  9. A new permanent cell line derived from the bank vole (Myodes glareolus) as cell culture model for zoonotic viruses

    PubMed Central

    2011-01-01

    Background Approximately 60% of emerging viruses are of zoonotic origin, with three-fourths derived from wild animals. Many of these zoonotic diseases are transmitted by rodents with important information about their reservoir dynamics and pathogenesis missing. One main reason for the gap in our knowledge is the lack of adequate cell culture systems as models for the investigation of rodent-borne (robo) viruses in vitro. Therefore we established and characterized a new cell line, BVK168, using the kidney of a bank vole, Myodes glareolus, the most abundant member of the Arvicolinae trapped in Germany. Results BVK168 proved to be of epithelial morphology expressing tight junctions as well as adherence junction proteins. The BVK168 cells were analyzed for their infectability by several arbo- and robo-viruses: Vesicular stomatitis virus, vaccinia virus, cowpox virus, Sindbis virus, Pixuna virus, Usutu virus, Inkoo virus, Puumalavirus, and Borna disease virus (BDV). The cell line was susceptible for all tested viruses, and most interestingly also for the difficult to propagate BDV. Conclusion In conclusion, the newly established cell line from wildlife rodents seems to be an excellent tool for the isolation and characterization of new rodent-associated viruses and may be used as in vitro-model to study properties and pathogenesis of these agents. PMID:21729307

  10. Initial site of synthesis of virus during rescue of simian virus 40 from heterokaryons of simian virus 40-transformed and susceptible cells.

    PubMed

    Wever, G H; Kit, S; Dubbs, D R

    1970-05-01

    Simian virus 40 (SV40) can be rescued from certain SV40-transformed hamster cells by fusion with susceptible African green monkey kidney (CV-1) cells, in the presence of ultraviolet-irradiated Sendai virus. We have determined the sites in which SV40 is produced during rescue in these heterokaryons. To determine the sequence, nuclei were isolated from fused cells at various times after fusion, separated on sucrose-density gradients, and assayed for infectious center formation and virus content on CV-1 monolayers. Virus was first detected in the transformed nucleus (40 hr postfusion), and later associated with both transformed and susceptible nuclei (68 to 72 hr). Viral rescue apparently does not depend upon the transfer of SV40 deoxyribonucleic acid to a susceptible CV-1 nucleus, since the transformed nucleus is the primary site of virus production. The time course of certain cytological events in the rescue process and in productive infection was found to be similar. PMID:4315957

  11. Hepatitis B virus antigens impair NK cell function.

    PubMed

    Yang, Yinli; Han, Qiuju; Zhang, Cai; Xiao, Min; Zhang, Jian

    2016-09-01

    An inadequate immune response of the host is thought to be a critical factor causing chronic hepatitis B virus (CHB) infection. Natural killer (NK) cells, as one of the key players in the eradication and control of viral infections, were functionally impaired in CHB patients, which might contribute to viral persistence. Here, we reported that HBV antigens HBsAg and HBeAg directly inhibited NK cell function. HBsAg and/or HBeAg blocked NK cell activation, cytokine production and cytotoxic granule release in human NK cell-line NK-92 cells, which might be related to the downregulation of activating receptors and upregulation of inhibitory receptor. Furthermore, the underlying mechanisms likely involved the suppression of STAT1, NF-κB and p38 MAPK pathways. These findings implicated that HBV antigen-mediated inhibition of NK cells might be an efficient strategy for HBV evasion, targeting the early antiviral responses mediated by NK cells and resulting in the establishment of chronic virus infection. Therefore, this study revealed the relationship between viral antigens and human immune function, especially a potential important interaction between HBV and innate immune responses. PMID:27341035

  12. Characterization of cell lines stably transfected with rubella virus replicons

    SciTech Connect

    Tzeng, Wen-Pin; Xu, Jie; Frey, Teryl K.

    2012-07-20

    Rubella virus (RUBV) replicons expressing a drug resistance gene and a gene of interest were used to select cell lines uniformly harboring the replicon. Replicons expressing GFP and a virus capsid protein GFP fusion (C-GFP) were compared. Vero or BHK cells transfected with either replicon survived drug selection and grew into a monolayer. However, survival was {approx}9-fold greater following transfection with the C-GFP-replicon than with the GFP-expressing replicon and while the C-GFP-replicon cells grew similarly to non-transfected cells, the GFP-replicon cells grew slower. Neither was due to the ability of the CP to enhance RNA synthesis but survival during drug selection was correlated with the ability of CP to inhibit apoptosis. Additionally, C-GFP-replicon cells were not cured of the replicon in the absence of drug selection. Interferon-alpha suppressed replicon RNA and protein synthesis, but did not cure the cells, explaining in part the ability of RUBV to establish persistent infections.

  13. Vaccinia virus strain differences in cell attachment and entry

    SciTech Connect

    Bengali, Zain; Townsley, Alan C.; Moss, Bernard

    2009-06-20

    Vaccinia virus (VACV) strain WR can enter cells by a low pH endosomal pathway or direct fusion with the plasma membrane at neutral pH. Here, we compared attachment and entry of five VACV strains in six cell lines and discovered two major patterns. Only WR exhibited pH 5-enhanced rate of entry following neutral pH adsorption to cells, which correlated with sensitivity to bafilomycin A1, an inhibitor of endosomal acidification. Entry of IHD-J, Copenhagen and Elstree strains were neither accelerated by pH 5 treatment nor prevented by bafilomycin A1. Entry of the Wyeth strain, although not augmented by pH 5, was inhibited by bafilomycin A1. WR and Wyeth were both relatively resistant to the negative effects of heparin on entry, whereas the other strains were extremely sensitive due to inhibition of cell binding. The relative sensitivities of individual vaccinia virus strains to heparin correlated inversely with their abilities to bind to and enter glycosaminoglycan-deficient sog9 cells but not other cell lines tested. These results suggested that that IHD-J, Copenhagen and Elstree have a more limited ability than WR and Wyeth to use the low pH endosomal pathway and are more dependent on binding to glycosaminoglycans for cell attachment.

  14. Reduced innate immune response, apoptosis, and virus release in cells cured of respiratory syncytial virus persistent infection.

    PubMed

    Herranz, Cristina; Melero, José A; Martínez, Isidoro

    2011-02-01

    It has been reported that cell clones isolated at different passages from a culture of HEp-2 cells infected persistently with human respiratory syncytial virus (HRSV) were cured of the virus. Further studies on one of these clones (31C1) are reported here, showing that 31C1 cells can still be infected by HRSV but release low amounts of virus to the culture supernatant, develop smaller and less numerous syncytia than the original HEp-2 cells, and display only a weak innate immune response to the infection. Accordingly, uninfected 31C1 cells, but not clones derived from uninfected HEp-2 cells, express low levels of TLR3 and RIG-I. In addition, 31C1 cells are partly resistant to apoptosis. These results indicate that persistent infection of HEp-2 cells by HRSV has selected cell variants, with changes affecting cell survival, virus growth and the innate immune response that may be valuable for studies of virus-cell interaction. PMID:21093006

  15. No detection of BK virus, JC virus, KI, WU and Merkel cell polyomaviruses in cerebrospinal fluid of patients with neurological complications after hematopoetic stem cell transplantation.

    PubMed

    Rubin, J; Giraud, G; Priftakis, P; Wide, K; Gustafsson, B; Ramqvist, T; Dalianis, T

    2011-10-01

    Neurological complications, often due to viral reactivation, after allogeneic hematopoetic stem cell transplantation (HSCT) are associated with increased mortality. Here, cerebrospinal fluid from 20 HSCT patients with neurological symptoms were analyzed and found to be negative by PCR for BK virus, JC virus, KI, WU and Merkel cell polyomavirus DNA. PMID:21965766

  16. Virus-Specific Messenger RNA and Nascent Polypeptides in Polyribosomes of Cells Replicating Murine Sarcoma-Leukemia Viruses

    PubMed Central

    Vecchio, G.; Tsuchida, N.; Shanmugam, G.; Green, M.

    1973-01-01

    We present evidence that virus-specific RNA is present in polyribosomes of transformed cells replicating the murine sarcoma-leukemia virus complex and that it serves as messenger RNA for the synthesis of viral-coded proteins. Both virus-specific RNA (detected by hybridization with the [3H]DNA product of the viral RNA-directed DNA polymerase) and nascent viral polypeptides (measured by precipitation with antiserum to purified virus) were found in membrane-bound and free polyribosomes. Membrane-bound polyribosomes contained a higher content of both virus-specific RNA and nascent viral polypeptides. From 60 to 70% of viral RNA sequences were released from polyribosomes with EDTA, consistent with a function as messenger RNA. Maximum amounts of both virus-specific RNA and nascent viral polypeptides were found in the polyribosome region sedimenting at about 350 S. PMID:4352969

  17. Oncolytic viruses & their specific targeting to tumour cells

    PubMed Central

    Singh, Prafull K.; Doley, Juwar; Kumar, G. Ravi; Sahoo, A.P.; Tiwari, Ashok K.

    2012-01-01

    Cancer is one of the major causes of death worldwide. In spite of achieving significant successes in medical sciences in the past few decades, the number of deaths due to cancer remains unchecked. The conventional chemotherapy and radiotherapy have limited therapeutic index and a plethora of treatment related side effects. This situation has provided an impetus for search of novel therapeutic strategies that can selectively destroy the tumour cells, leaving the normal cells unharmed. Viral oncotherapy is such a promising treatment modality that offers unique opportunity for tumour targeting. Numerous viruses with inherent anti-cancer activity have been identified and are in different phases of clinical trials. In the era of modern biotechnology and with better understanding of cancer biology and virology, it has become feasible to engineer the oncolytic viruses (OVs) to increase their tumour selectivity and enhance their oncolytic activity. In this review, the mechanisms by which oncolytic viruses kill the tumour cells have been discussed as also the development made in virotherapy for cancer treatment with emphasis on their tumour specific targeting. PMID:23168697

  18. PROVIDENCE VIRUS: A NEW MEMBER OF THE TETRAVIRIDAE THAT INFECTS CULTURED INSECT CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We identified a new member of the Tetraviridae, Providence virus (PrV), persistently infecting a cell line derived from the midgut of Helicoverpa zea (corn earworm). Virus purified from these cells also productively infected a H. zea fat body cell line and a cell line from whole embryos of the beet...

  19. Myxoma virus M063R is a host range gene essential for virus replication in rabbit cells.

    PubMed

    Barrett, John W; Shun Chang, Chew; Wang, Gen; Werden, Steven J; Shao, Zhuhong; Barrett, Catherine; Gao, Xiujuan; Belsito, Tara A; Villenevue, Danielle; McFadden, Grant

    2007-04-25

    The myxoma virus M063R gene product exhibits some sequence similarity to the poxvirus host range gene, C7L, of vaccinia virus. To address the potential host range function of the M063R gene product in rabbits, a deletion mutant of myxoma virus (vMyx63KO) was generated and characterized. vMyx63KO replicated to normal titre levels and produced foci that were indistinguishable from those produced by MV in vitro in a monkey kidney cell line (BGMK) that are permissive for wild type MV. However, vMyx63KO failed to replicate in all rabbit cell lines tested, including both primary and established cells lines, as well as cells derived from a variety of tissues. M063R expression was not required for myxoma virus binding, entry or early gene expression, whereas DNA replication was aborted and late genes were not expressed in vMyx63KO infected rabbit cells. Thus, the replication block for vMyx63KO in rabbit cells preceded the stage of late gene expression and DNA replication. Finally, an in vivo pathogenesis study indicated that vMyx63KO failed to cause any signs of classic myxomatosis in infected rabbits, but functioned as a non-replicating vaccine and provided protection for subsequent challenge by wild type myxoma virus. Altogether, these observations demonstrate that M063R plays a critical role in determining the host specificity of myxoma virus in rabbit cells. PMID:17184804

  20. Repertoire of virus-derived small RNAs produced by mosquito and mammalian cells in response to dengue virus infection.

    PubMed

    Schirtzinger, Erin E; Andrade, Christy C; Devitt, Nicholas; Ramaraj, Thiruvarangan; Jacobi, Jennifer L; Schilkey, Faye; Hanley, Kathryn A

    2015-02-01

    RNA interference (RNAi) is the major defense of many arthropods against arthropod-borne RNA viruses (arboviruses), but the role of RNAi in vertebrate immunity to arboviruses is not clear. RNA viruses can trigger RNAi in vertebrate cells, but the vertebrate interferon response may obscure this interaction. We quantified virus-derived small RNAs (vRNAs) generated by mosquito (U4.4) cells and interferon-deficient (Vero) and interferon-competent (HuH-7) mammalian cells infected with a single isolate of mosquito-borne dengue virus. Mosquito cells produced significantly more vRNAs than mammalian cells, and mosquito cell vRNAs were derived from both the positive- and negative-sense dengue genomes whereas mammalian cell vRNAs were derived primarily from positive-sense genome. Mosquito cell vRNAs were predominantly 21 nucleotides in length whereas mammalian cell vRNAs were between 12 and 36 nucleotides with a modest peak at 24 nucleotides. Hot-spots, regions of the virus genome that generated a disproportionate number of vRNAs, overlapped among the cell lines. PMID:25528416

  1. Infectious hematopoietic necrosis virus detected by separation and incubation of cells from salmonid cavity fluid.

    USGS Publications Warehouse

    Mulcahy, D.; Batts, W.N.

    1987-01-01

    Infectious hematopoietic necrosis (IHN) virus is usually detected by inoculating susceptible cell cultures with cavity ("ovarian") fluid (CF) from spawning females. We identified additional adult carriers of virus in spawning populations of steelhead trout (Salmo gairdneri) and sockeye salmon (Oncorhynchus nerka) by collecting nonerythrocytic cells from CF samples by low-speed centrifugation, culturing the cells for at least 7 d at 15 °C, and then testing the culture medium for virus. Virus appeared in the cultured cells from some samples of CF that remained negative during incubation. In additional samples of CF from these species, the virus titer increased in cultured cells compared with the titer in the original CF sample. With chinook salmon (O.tshawytscha), no negative samples converted to positive during incubation, but the virus titer was retained in incubated CF cells, but not in cell-free CF.

  2. Cross-reactive human B cell and T cell epitopes between influenza A and B viruses

    PubMed Central

    2013-01-01

    Influenza A and B viruses form different genera, which were originally distinguished by antigenic differences in their nucleoproteins and matrix 1 proteins. Cross-protection between these two genera has not been observed in animal experiments, which is consistent with the low homology in viral proteins common to both viruses except for one of three polymerase proteins, polymerase basic 1 (PB1). Recently, however, antibody and CD4+ T cell epitopes conserved between the two genera were identified in humans. A protective antibody epitope was located in the stalk region of the surface glycoprotein, hemagglutinin, and a CD4+ T cell epitope was located in the fusion peptide of the hemagglutinin. The fusion peptide was also found to contain antibody epitopes in humans and animals. A short stretch of well-conserved peptide was also identified in the other surface glycoprotein, neuraminidase, and antibodies binding to this peptide were generated by peptide immunization in rabbits. Although PB1, the only protein which has relatively high overall sequence homology between influenza A and B viruses, is not considered an immunodominant protein in the T cell responses to influenza A virus infection, amino acid sequence comparisons show that a considerable number of previously identified T cell epitopes in the PB1 of influenza A viruses are conserved in the PB1 of influenza B viruses. These data indicate that B and T cell cross-reactivity exists between influenza A and B viruses, which may have modulatory effects on the disease process and recovery. Although the antibody titers and the specific T cell frequencies induced by natural infection or standard vaccination may not be high enough to provide cross protection in humans, it might be possible to develop immunization strategies to induce these cross-reactive responses more efficiently. PMID:23886073

  3. Matricide and queen sex allocation in a yellowjacket wasp.

    PubMed

    Loope, Kevin J

    2016-08-01

    In many colonies of social insects, the workers compete with each other and with the queen over the production of the colony's males. In some species of social bees and wasps with annual societies, this intra-colony conflict even results in matricide-the killing of the colony's irreplaceable queen by a daughter worker. In colonies with low effective paternity and high worker-worker relatedness, workers value worker-laid males more than queen-laid males, and thus may benefit from queen killing. Workers gain by eliminating the queen because she is a competing source of male eggs and actively inhibits worker reproduction through policing. However, matricide may be costly to workers if it reduces the production of valuable new queens and workers. Here, I test a theoretical prediction regarding the timing of matricide in a wasp, Dolichovespula arenaria, recently shown to have facultative matricide based on intra-colony relatedness. Using analyses of collected, mature colonies and a surgical manipulation preventing queens from laying female eggs, I show that workers do not preferentially kill queens who are only producing male eggs. Instead, workers sometimes kill queens laying valuable females, suggesting a high cost of matricide. Although matricide is common and typically occurs only in low-paternity colonies, it seems that workers sometimes pay substantial costs in this expression of conflict over male parentage. PMID:27350328

  4. Matricide and queen sex allocation in a yellowjacket wasp

    NASA Astrophysics Data System (ADS)

    Loope, Kevin J.

    2016-08-01

    In many colonies of social insects, the workers compete with each other and with the queen over the production of the colony's males. In some species of social bees and wasps with annual societies, this intra-colony conflict even results in matricide—the killing of the colony's irreplaceable queen by a daughter worker. In colonies with low effective paternity and high worker-worker relatedness, workers value worker-laid males more than queen-laid males, and thus may benefit from queen killing. Workers gain by eliminating the queen because she is a competing source of male eggs and actively inhibits worker reproduction through policing. However, matricide may be costly to workers if it reduces the production of valuable new queens and workers. Here, I test a theoretical prediction regarding the timing of matricide in a wasp, Dolichovespula arenaria, recently shown to have facultative matricide based on intra-colony relatedness. Using analyses of collected, mature colonies and a surgical manipulation preventing queens from laying female eggs, I show that workers do not preferentially kill queens who are only producing male eggs. Instead, workers sometimes kill queens laying valuable females, suggesting a high cost of matricide. Although matricide is common and typically occurs only in low-paternity colonies, it seems that workers sometimes pay substantial costs in this expression of conflict over male parentage.

  5. Transformation of human cells by oncogenic viruses supports permissiveness for parvovirus H-1 propagation.

    PubMed Central

    Faisst, S; Schlehofer, J R; zur Hausen, H

    1989-01-01

    Parvovirus H-1 has been shown to suppress spontaneous and chemically or virally induced tumorigenesis in hamsters. In human cell culture systems propagation of H-1 is restricted to transformed cells, which are killed by H-1 infection, in contrast to normal diploid cells, which are nonpermissive for H-1. By analyzing the permissiveness of a variety of human cells for H-1, it was determined that the majority of tested transformed or immortalized cells which were permissive for H-1 contained the DNA of oncogenic viruses (human papillomavirus, simian virus 40, adenovirus, hepatitis B virus, Epstein-Barr virus, and human T-cell lymphotropic virus type I). Of six transformed cell lines negative for persisting tumor virus DNA, only two were permissive for H-1, while two were semipermissive and two were nonpermissive. Thus, persistence and expression of tumor virus functions appears to promote full permissiveness for H-1 in human cells. However, neither expression of genes of specific viral genomes nor the transformed state of apparently virus-free cells alone was sufficient to render human cells permissive for H-1. Therefore, the effect of tumor virus functions on H-1 in transformed cells seems to be indirect, probably mediated by cellular factors which are induced or switched off during the transformation process. It appears that similar factors are induced or switched off by 5-azacytidine or calcium phosphate, both known inducers of cellular gene expression. Images PMID:2495371

  6. Hepatitis C virus infection of cholangiocarcinoma cell lines.

    PubMed

    Fletcher, Nicola F; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K; van IJzendoorn, Sven C D; Baumert, Thomas F; Balfe, Peter; Afford, Simon; McKeating, Jane A

    2015-06-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  7. Hepatitis C virus infection of cholangiocarcinoma cell lines

    PubMed Central

    Fletcher, Nicola F.; Humphreys, Elizabeth; Jennings, Elliott; Osburn, William; Lissauer, Samantha; Wilson, Garrick K.; van IJzendoorn, Sven C. D.; Baumert, Thomas F.; Balfe, Peter; Afford, Simon

    2015-01-01

    Hepatitis C virus (HCV) infects the liver and hepatocytes are the major cell type supporting viral replication. Hepatocytes and cholangiocytes derive from a common hepatic progenitor cell that proliferates during inflammatory conditions, raising the possibility that cholangiocytes may support HCV replication and contribute to the hepatic reservoir. We screened cholangiocytes along with a panel of cholangiocarcinoma-derived cell lines for their ability to support HCV entry and replication. While primary cholangiocytes were refractory to infection and lacked expression of several entry factors, two cholangiocarcinoma lines, CC-LP-1 and Sk-ChA-1, supported efficient HCV entry; furthermore, Sk-ChA-1 cells supported full virus replication. In vivo cholangiocarcinomas expressed all of the essential HCV entry factors; however, cholangiocytes adjacent to the tumour and in normal tissue showed a similar pattern of receptor expression to ex vivo isolated cholangiocytes, lacking SR-BI expression, explaining their inability to support infection. This study provides the first report that HCV can infect cholangiocarcinoma cells and suggests that these heterogeneous tumours may provide a reservoir for HCV replication in vivo. PMID:25701818

  8. Live-Cell Imaging of Vaccinia Virus Recombination

    PubMed Central

    Paszkowski, Patrick; Noyce, Ryan S.; Evans, David H.

    2016-01-01

    Recombination between co-infecting poxviruses provides an important mechanism for generating the genetic diversity that underpins evolution. However, poxviruses replicate in membrane-bound cytoplasmic structures known as factories or virosomes. These are enclosed structures that could impede DNA mixing between co-infecting viruses, and mixing would seem to be essential for this process. We hypothesize that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses, and this requirement could delay or limit viral recombination. We have engineered vaccinia virus (VACV) to express overlapping portions of mCherry fluorescent protein fused to a cro DNA-binding element. In cells also expressing an EGFP-cro fusion protein, this permits live tracking of virus DNA and genetic recombination using confocal microscopy. Our studies show that different types of recombination events exhibit different timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants aren’t detected until well after late gene expression is well established. The delay supports the hypothesis that factories impede inter-viral recombination, but even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories. PMID:27525721

  9. Human papilloma virus, herpes simplex virus and epstein barr virus in oral squamous cell carcinoma from eight different countries.

    PubMed

    Jalouli, Jamshid; Jalouli, Miranda M; Sapkota, Dipak; Ibrahim, Salah O; Larsson, Per-Anders; Sand, Lars

    2012-02-01

    Oral squamous cell carcinoma (OSCC) is a major health problem in many parts of the world, and the major causative agents are thought to be the use of alcohol and tobacco. Oncogenic viruses have also been suggested to be involved in OSCC development. This study investigated the prevalence of human papillomaviruses (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) in 155 OSCC from eight different countries from different ethnic groups, continents and with different socioeconomic backgrounds. 41 A total of OSCCs were diagnosed in the tongue (26%) and 23 in the floor of the mouth (15%); the other 91 OSCCs were diagnosed in other locations (59%). The patients were also investigated regarding the use of alcohol and smoking and smokeless tobacco habits. Tissue samples were obtained from formalin-fixed, paraffin-embedded samples of the OSCC. DNA was extracted and the viral genome was examined by single, nested and semi-nested PCR assays. Sequencing of double-stranded DNA from the PCR product was carried out. Following sequencing of the HPV-, HSV- and EBV-positive PCR products, 100% homology between the sampels was found. Of all the 155 OSCCs examined, 85 (55%) were positive for EBV, 54 (35%) for HPV and 24 (15%) for HSV. The highest prevalence of HPV was seen in Sudan (65%), while HSV (55%) and EBV (80%) were most prevalent in the UK. In 34% (52/155) of all the samples examined, co-infection by two (46/155=30%) or three (6/155=4%) virus specimens was detected. The most frequent double infection was HPV with EBV in 21% (32/155) of all OSCCs. There was a statistically significant higher proportion of samples with HSV (p=0.026) and EBV (p=0.015) in industrialized countries (Sweden, Norway, UK and USA) as compared to developing countries (Sudan, India, Sri Lanka and Yemen). Furthermore, there was a statistically significant higher co-infection of HSV and EBV in samples from industrialized countries (p=0.00031). No firm conclusions could be drawn regarding the

  10. Functional Analysis of West Nile Virus Proteins in Human Cells.

    PubMed

    Kaufusi, Pakieli H; Tseng, Alanna; Nerurkar, Vivek R

    2016-01-01

    West Nile Virus (WNV) lineage 2 strains have been responsible for large outbreaks of neuroinvasive disease in the United States and Europe between 1999 and 2012. Different strains in this lineage have previously been shown to produce either severe or mild neuroinvasive disease in mice. Phylogenetic and amino acid comparisons between highly or less virulent lineage 2 strains have demonstrated that the nonstructural (NS) gene(s) were most variable. However, the roles of some of the NS proteins in virus life cycle are unknown. The aim of this chapter is to describe simple computational and experimental approaches that can be used to: (1) explore the possible roles of the NS proteins in virus life cycle and (2) test whether the subtle amino acid changes in WNV NS gene products contributed to the evolution of more virulent strains. The computational approaches include methods based on: (1) sequence similarity, (2) sequence motifs, and (3) protein membrane topology predictions. Highlighted experimental procedures include: (1) isolation of viral RNA from WNV-infected cells, (2) cDNA synthesis and PCR amplification of WNV genes, (3) cloning into GFP expression vector, (4) bacterial transformation, (5) plasmid isolation and purification, (6) transfection using activated dendrimers (Polyfect), and (7) immunofluorescence staining of transfected mammalian cells. PMID:27188549

  11. Tracking single viruses infecting their host cells using quantum dots.

    PubMed

    Liu, Shu-Lin; Wang, Zhi-Gang; Zhang, Zhi-Ling; Pang, Dai-Wen

    2016-03-01

    Single-virus tracking (SVT) technique, which uses microscopy to monitor the behaviors of viruses, is a vital tool to study the real-time and in situ infection dynamics and virus-related interactions in live cells. To make SVT a more versatile tool in biological research, the researchers have developed a quantum dot (QD)-based SVT technique, which can be utilized for long-term and highly sensitive tracking in live cells. In this review, we describe the development of a QD-based SVT technique and its biological applications. We first discuss the advantage of QDs as tags in the SVT field by comparing the conventional tags, and then focus on the implementation of QD-based SVT experiments, including the QD labeling strategy, instrumentation, and image analysis method. Next, we elaborate the recent advances of QD-based SVT in the biological field, and mainly emphasize the representative examples to show how to use this technique to acquire more meaningful biological information. PMID:26695711

  12. Detection of virus-specific RNA in simian sarcoma-leukemia virus-infected cells in in situ hybridization to viral complementary DNA.

    PubMed Central

    Kaufman, S L; Gallo, R C; Miller, N R

    1979-01-01

    An in situ molecular hybridization system which will detect retrovirus RNA in the cytoplasm of individual virus-infected cells has been developed. The technique was applied to cells infected with simian sarcoma-leukemia virus, where the virus-specific RNA was detected by hybridization to simian sarcoma-leukemia virus 3H-labeled complementary DNA. The system is useful for detecting viral RNA-containing cells in the presence of an excess of virus-negative cells and for determining which type of cell in a heterogenous population is expressing viral RNA. Images PMID:224220

  13. Role of natural killer cells in innate protection against lethal ebola virus infection.

    PubMed

    Warfield, Kelly L; Perkins, Jeremy G; Swenson, Dana L; Deal, Emily M; Bosio, Catharine M; Aman, M Javad; Yokoyama, Wayne M; Young, Howard A; Bavari, Sina

    2004-07-19

    Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1-3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell-mediated protection clearly depended on perforin, but not interferon-gamma secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection. PMID:15249592

  14. Human Vγ9Vδ2-T cells efficiently kill influenza virus-infected lung alveolar epithelial cells

    PubMed Central

    Li, Hong; Xiang, Zheng; Feng, Ting; Li, Jinrong; Liu, Yinping; Fan, Yingying; Lu, Qiao; Yin, Zhongwei; Yu, Meixing; Shen, Chongyang; Tu, Wenwei

    2013-01-01

    γδ-T cells play an indispensable role in host defense against different viruses, including influenza A virus. However, whether these cells have cytotoxic activity against influenza virus-infected lung alveolar epithelial cells and subsequently contribute to virus clearance remains unknown. Using influenza virus-infected A549 cells, human lung alveolar epithelial cells, we investigated the cytotoxic activity of aminobisphosphonate pamidronate (PAM)-expanded human Vγ9Vδ2-T cells and their underlying mechanisms. We found that PAM could selectively activate and expand human Vγ9Vδ2-T cells. PAM-expanded human Vγ9Vδ2-T cells efficiently killed influenza virus-infected lung alveolar epithelial cells and inhibited virus replication. The cytotoxic activity of PAM-expanded Vγ9Vδ2-T cells was dependent on cell-to-cell contact and required NKG2D activation. Perforin–granzyme B, tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas–Fas ligand (FasL) pathways were involved in their cytotoxicity. Our study suggests that targeting γδ-T cells by PAM can potentially offer an alternative option for the treatment of influenza virus. PMID:23353835

  15. On the origin of cells and viruses: primordial virus world scenario.

    PubMed

    Koonin, Eugene V

    2009-10-01

    It is proposed that the precellular stage of biological evolution unraveled within networks of inorganic compartments that harbored a diverse mix of virus-like genetic elements. This stage of evolution might makes up the Last Universal Cellular Ancestor (LUCA) that more appropriately could be denoted Last Universal Cellular Ancestral State (LUCAS). Such a scenario recapitulates the ideas of J. B. S. Haldane sketched in his classic 1928 essay. However, unlike in Haldane's day, considerable support for this scenario exits today: lack of homology between core DNA replication system components in archaea and bacteria, distinct membrane chemistries and enzymes of lipid biosynthesis in archaea and bacteria, spread of several viral hallmark genes among diverse groups of viruses, and the extant archaeal and bacterial chromosomes appear to be shaped by accretion of diverse, smaller replicons. Under the viral model of precellular evolution, the key components of cells originated as components of virus-like entities. The two surviving types of cellular life forms, archaea and bacteria, might have emerged from the LUCAS independently, along with, probably, numerous forms now extinct. PMID:19845627

  16. Influenza A Virus Polymerase Is a Site for Adaptive Changes during Experimental Evolution in Bat Cells

    PubMed Central

    Poole, Daniel S.; Yú, Shuǐqìng; Caì, Yíngyún; Dinis, Jorge M.; Müller, Marcel A.; Jordan, Ingo; Friedrich, Thomas C.; Kuhn, Jens H.

    2014-01-01

    ABSTRACT The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and

  17. Human Papilloma Virus and Squamous Cell Carcinoma of the Anus

    PubMed Central

    Gami, Bhavna; Kubba, Faris; Ziprin, Paul

    2014-01-01

    The incidence of anal cancer is increasing. In the UK, the incidence is estimated at approximately 1.5 per 100,000. Most of this increase is attributed to certain at-risk populations. Persons who are human immunodeficiency virus (HIV)–positive and men who have sex with men (MSM), Organ transplant recipients, women with a history of cervical cancer, human papilloma virus (HPV), or cervical intraepithelial neoplasia (CIN) are known to have a greater risk for anal cancer. This paper will focus on HPV as a risk factor for anal intraepithelial neoplasia (AIN) and discusses the etiology, anatomy, pathogenesis, management of squamous cell carcinoma (SCC) of the anus. PMID:25288893

  18. In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission.

    PubMed

    Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

    2015-11-01

    Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell-cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105-106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT-BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies. PMID:26569290

  19. Dengue Virus Infection of Mast Cells Triggers Endothelial Cell Activation ▿

    PubMed Central

    Brown, Michael G.; Hermann, Laura L.; Issekutz, Andrew C.; Marshall, Jean S.; Rowter, Derek; Al-Afif, Ayham; Anderson, Robert

    2011-01-01

    Vascular perturbation is a hallmark of severe forms of dengue disease. We show here that antibody-enhanced dengue virus infection of primary human cord blood-derived mast cells (CBMCs) and the human mast cell-like line HMC-1 results in the release of factor(s) which activate human endothelial cells, as evidenced by increased expression of the adhesion molecules ICAM-1 and VCAM-1. Endothelial cell activation was prevented by pretreatment of mast cell-derived supernatants with a tumor necrosis factor (TNF)-specific blocking antibody, thus identifying TNF as the endothelial cell-activating factor. Our findings suggest that mast cells may represent an important source of TNF, promoting vascular endothelial perturbation following antibody-enhanced dengue virus infection. PMID:21068256

  20. Adoptive Immunotherapy using Regulatory T cells and Virus-specific T cells Derived from Cord Blood

    PubMed Central

    Hanley, Patrick J.; Bollard, Catherine M.; Brunstein, Claudio G

    2014-01-01

    Cord blood transplantation, an alternative to traditional stem cell transplants (bone marrow or peripheral blood stem cell transplantation), is an attractive option for patients lacking suitable stem cell transplant donors. Cord blood units have also proven to be a valuable donor source for the development of cellular therapeutics. Virus-specific T cells and regulatory T cells are two cord blood derived products that have shown promise in early phase clinical trials to prevent and/or treat viral infections and graft-versus-host disease (GvHD), respectively. Here we describe how current strategies utilizing cord blood-derived regulatory T cells and virus-specific T cells have been developed to improve outcomes for cord blood transplant recipients. PMID:25632003

  1. Induction of Cytopathogenicity in Human Glioblastoma Cells by Chikungunya Virus

    PubMed Central

    Abraham, Rachy; Mudaliar, Prashant; Padmanabhan, Aiswaria; Sreekumar, Easwaran

    2013-01-01

    Chikungunya virus (CHIKV), an arthritogenic old-world alphavirus, has been implicated in the central nervous system (CNS) infection in infants and elderly patients. Astrocytes are the major immune cells of the brain parenchyma that mediate inflammation. In the present study we found that a local isolate of CHIKV infect and activate U-87 MG cells, a glioblastoma cell line of human astrocyte origin. The infection kinetics were similar in infected U-87 MG cells and the human embryo kidney (HEK293) cells as indicated by immunofluorescence and plaque assays, 24h post-infection (p.i.). In infected U-87 MG cells, apoptosis was detectable from 48h p.i. evidenced by DNA fragmentation, PARP cleavage, loss of mitochondrial membrane potential, nuclear condensation and visible cytopathic effects in a dose and time-dependent manner. XBP1 mRNA splicing and eIF2α phosphorylation studies indicated the occurrence of endoplasmic reticulum stress in infected cells. In U-87 MG cells stably expressing a green fluorescent protein-tagged light chain-3 (GFP-LC3) protein, CHIKV infection showed increased autophagy response. The infection led to an enhanced expression of the mRNA transcripts of the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and CXCL9 within 24h p.i. Significant up-regulation of the proteins of RIG-I like receptor (RLR) pathway, such as RIG-I and TRAF-6, was observed indicating the activation of the cytoplasmic-cellular innate immune response. The overall results show that the U-87 MG cell line is a potential in vitro model for in depth study of these molecular pathways in response to CHIKV infection. The responses in these cells of CNS origin, which are inherently defective in Type I interferon response, could be analogous to that occurring in infants and very old patients who also have a compromised interferon-response. The results also point to the intriguing possibility of using this virus for studies to develop oncolytic virus therapy approaches against

  2. Induction of cytopathogenicity in human glioblastoma cells by chikungunya virus.

    PubMed

    Abraham, Rachy; Mudaliar, Prashant; Padmanabhan, Aiswaria; Sreekumar, Easwaran

    2013-01-01

    Chikungunya virus (CHIKV), an arthritogenic old-world alphavirus, has been implicated in the central nervous system (CNS) infection in infants and elderly patients. Astrocytes are the major immune cells of the brain parenchyma that mediate inflammation. In the present study we found that a local isolate of CHIKV infect and activate U-87 MG cells, a glioblastoma cell line of human astrocyte origin. The infection kinetics were similar in infected U-87 MG cells and the human embryo kidney (HEK293) cells as indicated by immunofluorescence and plaque assays, 24h post-infection (p.i.). In infected U-87 MG cells, apoptosis was detectable from 48h p.i. evidenced by DNA fragmentation, PARP cleavage, loss of mitochondrial membrane potential, nuclear condensation and visible cytopathic effects in a dose and time-dependent manner. XBP1 mRNA splicing and eIF2α phosphorylation studies indicated the occurrence of endoplasmic reticulum stress in infected cells. In U-87 MG cells stably expressing a green fluorescent protein-tagged light chain-3 (GFP-LC3) protein, CHIKV infection showed increased autophagy response. The infection led to an enhanced expression of the mRNA transcripts of the pro-inflammatory cytokines IL-1β, TNF-α, IL-6 and CXCL9 within 24h p.i. Significant up-regulation of the proteins of RIG-I like receptor (RLR) pathway, such as RIG-I and TRAF-6, was observed indicating the activation of the cytoplasmic-cellular innate immune response. The overall results show that the U-87 MG cell line is a potential in vitro model for in depth study of these molecular pathways in response to CHIKV infection. The responses in these cells of CNS origin, which are inherently defective in Type I interferon response, could be analogous to that occurring in infants and very old patients who also have a compromised interferon-response. The results also point to the intriguing possibility of using this virus for studies to develop oncolytic virus therapy approaches against

  3. Seasonal Dynamics in the Chemistry and Structure of the Fat Bodies of Bumblebee Queens.

    PubMed

    Votavová, Alena; Tomčala, Aleš; Kofroňová, Edita; Kudzejová, Michaela; Šobotník, Jan; Jiroš, Pavel; Komzáková, Olga; Valterová, Irena

    2015-01-01

    Insects' fat bodies are responsible for nutrient storage and for a significant part of intermediary metabolism. Thus, it can be expected that the structure and content of the fat body will adaptively change, if an insect is going through different life stages. Bumblebee queens belong to such insects as they dramatically change their physiology several times over their lives in relation to their solitary overwintering, independent colony foundation stage, and during the colony life-cycle ending in the senescent stage. Here, we report on changes in the ultrastructure and lipid composition of the peripheral fat body of Bombus terrestris queens in relation to seasonal changes in the queens' activity. Six life stages are defined and evaluated in particular: pharate, callow, before and after hibernation, egg-laying, and senescence. Transmission electron microscopy revealed that the fat body contained two main cell types-adipocytes and oenocytes. Only adipocytes reveal important changes related to the life phase, and mostly the ration between inclusion and cytoplasm volume varies among particular stages. Both electron microscopy and chemical analyses of lipids highlighted seasonal variability in the quantity of the stored lipids, which peaked prior to hibernation. Triacylglycerols appeared to be the main energy source during hibernation, while the amount of glycogen before and after hibernation remained unchanged. In addition, we observed that the representation of some fatty acids within the triacylglycerols change during the queen's life. Last but not least, we show that fat body cell membranes do not undergo substantial changes concerning phospholipid composition in relation to overwintering. This finding supports the hypothesis that the cold-adaptation strategy of bumblebee queens is more likely to be based on polyol accumulation than on the restructuring of lipid membranes. PMID:26559946

  4. Biology of Zika Virus Infection in Human Skin Cells

    PubMed Central

    Hamel, Rodolphe; Dejarnac, Ophélie; Wichit, Sineewanlaya; Ekchariyawat, Peeraya; Neyret, Aymeric; Luplertlop, Natthanej; Perera-Lecoin, Manuel; Surasombatpattana, Pornapat; Talignani, Loïc; Thomas, Frédéric; Cao-Lormeau, Van-Mai; Choumet, Valérie; Briant, Laurence; Desprès, Philippe; Amara, Ali; Yssel, Hans

    2015-01-01

    ABSTRACT Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune

  5. Potential role of natural killer cells in controlling tumorigenesis by human T-cell leukemia viruses.

    PubMed Central

    Feuer, G; Stewart, S A; Baird, S M; Lee, F; Feuer, R; Chen, I S

    1995-01-01

    Human T-cell leukemia virus (HTLV) is the etiologic agent of adult T-cell leukemia (ATL), a malignancy of T lymphocytes that is characterized by a long latency period after virus exposure. Intraperitoneal inoculation of severe combined immunodeficient (SCID) mice with HTLV-transformed cell lines and ATL tumor cells was employed to investigate the tumorigenic potential of HTLV type I (HTLV-I)-infected cells. In contrast to inoculation of ATL (RV-ATL) cells into SCID mice, which resulted in the formation of lymphomas, inoculation of HTLV-I- and HTLV-II-transformed cell lines (SLB-I and JLB-II cells, respectively) did not result in tumor formation. Immunosuppression of SCID mice, either by whole-body irradiation or by treatment with an antiserum, anti-asialo GM1 (alpha-AGM1), which transiently abrogates natural killer cell activity in vivo, was necessary to establish the growth of tumors derived from HTLV-transformed cell lines. PCR and flow cytometric studies reveal that HTLV-I-transformed cells are eliminated from the peritoneal cavities of inoculated mice by 3 days postinoculation; in contrast, RV-ATL cells persist and are detected until the mice succumb to lymphoma development. The differing behaviors of HTLV-infected cell lines and ATL tumor cells in SCID mice suggest that ATL cells have a higher tumorigenic potential in vivo than do HTLV-infected cell lines because of their ability to evade natural killer cell-mediated cytolysis. PMID:7815516

  6. Honeybee colony disorder in crop areas: the role of pesticides and viruses.

    PubMed

    Simon-Delso, Noa; San Martin, Gilles; Bruneau, Etienne; Minsart, Laure-Anne; Mouret, Coralie; Hautier, Louis

    2014-01-01

    As in many other locations in the world, honeybee colony losses and disorders have increased in Belgium. Some of the symptoms observed rest unspecific and their causes remain unknown. The present study aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Honeybees, wax, beebread and honey samples were collected. Morbidity and mortality information provided by beekeepers, colony clinical visits and availability of analytical matrix were used to form 2 groups: healthy colonies and colonies with disorders (n = 29, n = 25, respectively). Disorders included: (1) dead colonies or colonies in which part of the colony appeared dead, or had disappeared; (2) weak colonies; (3) queen loss; (4) problems linked to brood and not related to any known disease. Five common viruses and 99 pesticides (41 fungicides, 39 insecticides and synergist, 14 herbicides, 5 acaricides and metabolites) were quantified in the samples.The main symptoms observed in the group with disorders are linked to brood and queens. The viruses most frequently found are Black Queen Cell Virus, Sac Brood Virus, Deformed Wing Virus. No significant difference in virus load was observed between the two groups. Three acaricides, 5 insecticides and 13 fungicides were detected in the analysed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to our results, the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies. PMID:25048715

  7. Honeybee Colony Disorder in Crop Areas: The Role of Pesticides and Viruses

    PubMed Central

    Simon-Delso, Noa; San Martin, Gilles; Bruneau, Etienne; Minsart, Laure-Anne; Mouret, Coralie; Hautier, Louis

    2014-01-01

    As in many other locations in the world, honeybee colony losses and disorders have increased in Belgium. Some of the symptoms observed rest unspecific and their causes remain unknown. The present study aims to determine the role of both pesticide exposure and virus load on the appraisal of unexplained honeybee colony disorders in field conditions. From July 2011 to May 2012, 330 colonies were monitored. Honeybees, wax, beebread and honey samples were collected. Morbidity and mortality information provided by beekeepers, colony clinical visits and availability of analytical matrix were used to form 2 groups: healthy colonies and colonies with disorders (n = 29, n = 25, respectively). Disorders included: (1) dead colonies or colonies in which part of the colony appeared dead, or had disappeared; (2) weak colonies; (3) queen loss; (4) problems linked to brood and not related to any known disease. Five common viruses and 99 pesticides (41 fungicides, 39 insecticides and synergist, 14 herbicides, 5 acaricides and metabolites) were quantified in the samples.The main symptoms observed in the group with disorders are linked to brood and queens. The viruses most frequently found are Black Queen Cell Virus, Sac Brood Virus, Deformed Wing Virus. No significant difference in virus load was observed between the two groups. Three acaricides, 5 insecticides and 13 fungicides were detected in the analysed samples. A significant correlation was found between the presence of fungicide residues and honeybee colony disorders. A significant positive link could also be established between the observation of disorder and the abundance of crop surface around the beehive. According to our results, the role of fungicides as a potential stressor for honeybee colonies should be further studied, either by their direct and/or indirect impacts on bees and bee colonies. PMID:25048715

  8. Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound.

    PubMed

    Okunaga, Shusuke; Takasu, Ayako; Meshii, Noritoshi; Imai, Tomoaki; Hamada, Masakagu; Iwai, Soichi; Yura, Yoshiaki

    2015-10-01

    Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the 134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval. PMID:26516901

  9. Entry of Oncolytic Herpes Simplex Virus into Human Squamous Cell Carcinoma Cells by Ultrasound

    PubMed Central

    Okunaga, Shusuke; Takasu, Ayako; Meshii, Noritoshi; Imai, Tomoaki; Hamada, Masakagu; Iwai, Soichi; Yura, Yoshiaki

    2015-01-01

    Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporations, on the cell membrane. Whether oncolytic herpes simplex virus type 1 (HSV-1) can be introduced into oral squamous cell carcinoma (SCC) cells through membrane pores remains undetermined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the γ134.5 gene and fusogenic, were used. Cells were exposed to ultrasound in the presence or absence of microbubbles. The increase of virus entry was estimated by plaque numbers. Viral infection was hardly established without the adsorption step, but plaque number was increased by the exposure of HSV-1-inoculated cells to ultrasound. Plaque number was also increased even if SAS cells were exposed to ultrasound and inoculated with RH2 without the adsorption step. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed depressed spots on the cell surface after exposure to ultrasound. These results suggest that oncolytic HSV-1 RH2 can be introduced into SAS cells through ultrasound-mediated pores of the cell membrane that are resealed after an interval. PMID:26516901

  10. Curcumin Inhibits Rift Valley Fever Virus Replication in Human Cells*

    PubMed Central

    Narayanan, Aarthi; Kehn-Hall, Kylene; Senina, Svetlana; Lundberg, Lindsay; Van Duyne, Rachel; Guendel, Irene; Das, Ravi; Baer, Alan; Bethel, Laura; Turell, Michael; Hartman, Amy Lynn; Das, Bhaskar; Bailey, Charles; Kashanchi, Fatah

    2012-01-01

    Rift Valley fever virus (RVFV) is an arbovirus that is classified as a select agent, an emerging infectious virus, and an agricultural pathogen. Understanding RVFV-host interactions is imperative to the design of novel therapeutics. Here, we report that an infection by the MP-12 strain of RVFV induces phosphorylation of the p65 component of the NFκB cascade. We demonstrate that phosphorylation of p65 (serine 536) involves phosphorylation of IκBα and occurs through the classical NFκB cascade. A unique, low molecular weight complex of the IKK-β subunit can be observed in MP-12-infected cells, which we have labeled IKK-β2. The IKK-β2 complex retains kinase activity and phosphorylates an IκBα substrate. Inhibition of the IKK complex using inhibitors impairs viral replication, thus alluding to the requirement of an active IKK complex to the viral life cycle. Curcumin strongly down-regulates levels of extracellular infectious virus. Our data demonstrated that curcumin binds to and inhibits kinase activity of the IKK-β2 complex in infected cells. Curcumin partially exerts its inhibitory influence on RVFV replication by interfering with IKK-β2-mediated phosphorylation of the viral protein NSs and by altering the cell cycle of treated cells. Curcumin also demonstrated efficacy against ZH501, the fully virulent version of RVFV. Curcumin treatment down-regulated viral replication in the liver of infected animals. Our data point to the possibility that RVFV infection may result in the generation of novel versions of host components (such as IKK-β2) that, by virtue of altered protein interaction and function, qualify as unique therapeutic targets. PMID:22847000

  11. B cell fate decisions following influenza virus infection

    PubMed Central

    Rothaeusler, Kristina; Baumgarth, Nicole

    2010-01-01

    Summary Rapidly induced, specific antibodies generated in extrafollicular foci are important components of early immune protection to influenza virus. The signal(s) that prompt B cells to participate in extrafollicular rather than germinal center responses are incompletely understood. To study the regulation of early B cell differentiation events following influenza infection, we exploited earlier findings of a strong contribution of C12 idiotype-expressing B cells to the primary hemagglutinin (HA)-specific response against influenza A/PR/8/34. Using an idiotype-specific mAb to C12 and labeled-HA, in conjunction with multicolor flow cytometry, we followed the fate of C12Id-expressing influenza HA-specific B cells in wildtype BALB/c mice, requiring neither genetic manipulation nor adoptive cell transfer. Our studies demonstrate that HA-specific C12Id+ B cells are phenotypically indistinguishable from follicular B cells. While they induced both extrafollicular and germinal center responses, extrafollicular responses were strongly predominant. Provision of increased HA-specific T cell help increased the magnitude of the extrafollicular response, but did not shift the C12Id+ response towards germinal center formation. Collectively the data are consistent with the hypothesis that B cell fate-determination following activation is a stochastic process in which infection-induced innate signals might drive the preferential expansion of the early extrafollicular response. PMID:19946883

  12. Glycoprotein E of Varicella-Zoster Virus Enhances Cell-Cell Contact in Polarized Epithelial Cells

    PubMed Central

    Mo, Chengjun; Schneeberger, Eveline E.; Arvin, Ann M.

    2000-01-01

    Varicella-zoster virus (VZV) infection involves the cell-cell spread of virions, but how viral proteins interact with the host cell membranes that comprise intercellular junctions is not known. Madin-Darby canine kidney (MDCK) cells were constructed to express the glycoproteins gE, gI, or gE/gI constitutively and were used to examine the effects of these VZV glycoproteins in polarized epithelial cells. At low cell density, VZV gE induced partial tight junction (TJ) formation under low-calcium conditions, whether expressed alone or with gI. Although most VZV gE was intracellular, gE was also shown to colocalize with the TJ protein ZO-1 with or without concomitant expression of gI. Freeze fracture electron microscopy revealed normal TJ strand morphology in gE-expressing MDCK cells. Functionally, the expression of gE was associated with a marked acceleration in the establishment of maximum transepithelial electrical resistance (TER) in MDCK-gE cells; MDCK-gI and MDCK-gE/gI cells exhibited a similar pattern of early TER compared to MDCK cells, although peak resistances were lower than those of gE alone. VZV gE expression altered F-actin organization and lipid distribution, but coexpression of gI modulated these effects. Two regions of the gE ectodomain, amino acids (aa) 278 to 355 and aa 467 to 498, although lacking Ca2+ binding motifs, exhibit similarities with corresponding regions of the cell adhesion molecules, E-cadherin and desmocollin. These observations suggest that VZV gE and gE/gI may contribute to viral pathogenesis by facilitating epithelial cell-cell contacts. PMID:11070038

  13. In Vitro Evolution of Bovine Foamy Virus Variants with Enhanced Cell-Free Virus Titers and Transmission

    PubMed Central

    Bao, Qiuying; Hipp, Michaela; Hugo, Annette; Lei, Janet; Liu, Yang; Kehl, Timo; Hechler, Torsten; Löchelt, Martin

    2015-01-01

    Virus transmission is essential for spreading viral infections and is a highly coordinated process which occurs by cell-free transmission or cell–cell contact. The transmission of Bovine Foamy Virus (BFV) is highly cell-associated, with undetectable cell-free transmission. However, BFV particle budding can be induced by overexpression of wild-type (wt) BFV Gag and Env or artificial retargeting of Gag to the plasma membrane via myristoylation membrane targeting signals, closely resembling observations in other foamy viruses. Thus, the particle release machinery of wt BFV appears to be an excellent model system to study viral adaption to cell-free transmission by in vitro selection and evolution. Using selection for BFV variants with high cell-free infectivity in bovine and non-bovine cells, infectivity dramatically increased from almost no infectious units to about 105–106 FFU (fluorescent focus forming units)/mL in both cell types. Importantly, the selected BFV variants with high titer (HT) cell-free infectivity could still transmit via cell-cell contacts and were neutralized by serum from naturally infected cows. These selected HT–BFV variants will shed light into virus transmission and potential routes of intervention in the spread of viral infections. It will also allow the improvement or development of new promising approaches for antiretroviral therapies. PMID:26569290

  14. Three mechanisms of Red Queen dynamics

    PubMed Central

    Khibnik, A. I.; Kondrashov, A. S.

    1997-01-01

    Models describing systems of coevolving populations often have asymptotically non-equilibrium dynamics (Red Queen dynamics (RQD)). We claim that if evolution is much slower than ecological changes, RQD arises due to either fast ecological processes, slow genetical processes, or to their interaction. The three corresponding generic types of RQD can be studied using singular perturbation theory and have very different properties and biological implications. We present simple examples of ecological, genetical, and ecogenetical RQD and describe how they may be recognized in natural populations. In particular, ecogenetical RQD often involve alternations of long epochs with radically different dynamics.

  15. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    SciTech Connect

    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-10-25

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  16. Retrovirus gene expression during the cell cycle. I. Virus production, synthesis, and expression of viral proteins in Rauscher murine leukemia virus-infected mouse cells.

    PubMed Central

    Balazs, I; Caldarella, J

    1981-01-01

    Synchronized mouse cells (JLS-V9) chronically infected with Rauscher murine leukemia virus were used to study virus production, the synthesis of gag and env precursor proteins, and the expression of env protein on the cell surface during the cell cycle. The amount of virus released into the medium by synchronized cells during a 30-min interval was determined by using the XC plaque assay and by measuring reverse transcriptase activity. The results show that virus production occurs during mitosis. Labeling of the cell surface of synchronized cells with 125I or with fluorescein-conjugated antiserum shows that the amount of gp 70env on the cell surface parallels cellular growth. Therefore, the cell cycle-dependent release of virus is not accompanied by similar variations in the amount of viral envelope protein on the cell surface. Immunoprecipitation of cells labeled with [35S]methionine, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was used to measure viral protein synthesis during the cell cycle. The rate of synthesis of gag precursor proteins show three maximums corresponding to the G1, middle S, and late S to G2 phases of the cell cycle. The rate of synthesis of env precursor proteins does not change, suggesting that in these cells the synthesis of these two gene products is controlled separately. Images PMID:7288918

  17. A plasmid-based reporter system for live cell imaging of dengue virus infected cells.

    PubMed

    Medin, Carey L; Valois, Sierra; Patkar, Chinmay G; Rothman, Alan L

    2015-01-01

    Cell culture models are used widely to study the effects of dengue virus (DENV) on host cell function. Current methods of identification of cells infected with an unmodified DENV requires fixation and permeablization of cells to allow DENV-specific antibody staining. This method does not permit imaging of viable cells over time. In this report, a plasmid-based reporter was developed to allow non-destructive identification of DENV-infected cells. The plasmid-based reporter was demonstrated to be broadly applicable to the four DENV serotypes, including low-passaged strains, and was specifically cleaved by the viral protease with minimal interference on viral production. This study reveals the potential for this novel reporter system to advance the studies of virus-host interactions during DENV infection. PMID:25445884

  18. Evidences Suggesting Involvement of Viruses in Oral Squamous Cell Carcinoma

    PubMed Central

    Gupta, Kanupriya; Metgud, Rashmi

    2013-01-01

    Oral cancer is one of the most common cancers and it constitutes a major health problem particularly in developing countries. Oral squamous cell carcinoma (OSCC) represents the most frequent of all oral neoplasms. Several risk factors have been well characterized to be associated with OSCC with substantial evidences. The etiology of OSCC is complex and involves many factors. The most clearly defined potential factors are smoking and alcohol, which substantially increase the risk of OSCC. However, despite this clear association, a substantial proportion of patients develop OSCC without exposure to them, emphasizing the role of other risk factors such as genetic susceptibility and oncogenic viruses. Some viruses are strongly associated with OSCC while the association of others is less frequent and may depend on cofactors for their carcinogenic effects. Therefore, the exact role of viruses must be evaluated with care in order to improve the diagnosis and treatment of OSCC. Although a viral association within a subset of OSCC has been shown, the molecular and histopathological characteristics of these tumors have yet to be clearly defined. PMID:24455418

  19. Nonreplicating Vaccinia Virus Vectors Expressing the H5 Influenza Virus Hemagglutinin Produced in Modified Vero Cells Induce Robust Protection▿

    PubMed Central

    Mayrhofer, Josef; Coulibaly, Sogue; Hessel, Annett; Holzer, Georg W.; Schwendinger, Michael; Brühl, Peter; Gerencer, Marijan; Crowe, Brian A.; Shuo, Shen; Hong, Wanjing; Tan, Yee Joo; Dietrich, Barbara; Sabarth, Nicolas; Savidis-Dacho, Helga; Kistner, Otfried; Barrett, P. Noel; Falkner, Falko G.

    2009-01-01

    The timely development of safe and effective vaccines against avian influenza virus of the H5N1 subtype will be of the utmost importance in the event of a pandemic. Our aim was first to develop a safe live vaccine which induces both humoral and cell-mediated immune responses against human H5N1 influenza viruses and second, since the supply of embryonated eggs for traditional influenza vaccine production may be endangered in a pandemic, an egg-independent production procedure based on a permanent cell line. In the present article, the generation of a complementing Vero cell line suitable for the production of safe poxviral vaccines is described. This cell line was used to produce a replication-deficient vaccinia virus vector H5N1 live vaccine, dVV-HA5, expressing the hemagglutinin of a virulent clade 1 H5N1 strain. This experimental vaccine was compared with a formalin-inactivated whole-virus vaccine based on the same clade and with different replicating poxvirus-vectored vaccines. Mice were immunized to assess protective immunity after high-dose challenge with the highly virulent A/Vietnam/1203/2004(H5N1) strain. A single dose of the defective live vaccine induced complete protection from lethal homologous virus challenge and also full cross-protection against clade 0 and 2 challenge viruses. Neutralizing antibody levels were comparable to those induced by the inactivated vaccine. Unlike the whole-virus vaccine, the dVV-HA5 vaccine induced substantial amounts of gamma interferon-secreting CD8 T cells. Thus, the nonreplicating recombinant vaccinia virus vectors are promising vaccine candidates that induce a broad immune response and can be produced in an egg-independent and adjuvant-independent manner in a proven vector system. PMID:19279103

  20. Ectopic Expression of Vaccinia Virus E3 and K3 Cannot Rescue Ectromelia Virus Replication in Rabbit RK13 Cells

    PubMed Central

    Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R.

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  1. Ectopic expression of vaccinia virus E3 and K3 cannot rescue ectromelia virus replication in rabbit RK13 cells.

    PubMed

    Hand, Erin S; Haller, Sherry L; Peng, Chen; Rothenburg, Stefan; Hersperger, Adam R

    2015-01-01

    As a group, poxviruses have been shown to infect a wide variety of animal species. However, there is individual variability in the range of species able to be productively infected. In this study, we observed that ectromelia virus (ECTV) does not replicate efficiently in cultured rabbit RK13 cells. Conversely, vaccinia virus (VACV) replicates well in these cells. Upon infection of RK13 cells, the replication cycle of ECTV is abortive in nature, resulting in a greatly reduced ability to spread among cells in culture. We observed ample levels of early gene expression but reduced detection of virus factories and severely blunted production of enveloped virus at the cell surface. This work focused on two important host range genes, named E3L and K3L, in VACV. Both VACV and ECTV express a functional protein product from the E3L gene, but only VACV contains an intact K3L gene. To better understand the discrepancy in replication capacity of these viruses, we examined the ability of ECTV to replicate in wild-type RK13 cells compared to cells that constitutively express E3 and K3 from VACV. The role these proteins play in the ability of VACV to replicate in RK13 cells was also analyzed to determine their individual contribution to viral replication and PKR activation. Since E3L and K3L are two relevant host range genes, we hypothesized that expression of one or both of them may have a positive impact on the ability of ECTV to replicate in RK13 cells. Using various methods to assess virus growth, we did not detect any significant differences with respect to the replication of ECTV between wild-type RK13 compared to versions of this cell line that stably expressed VACV E3 alone or in combination with K3. Therefore, there remain unanswered questions related to the factors that limit the host range of ECTV. PMID:25734776

  2. Alternative mating behaviors of the queen polymorphic ant Temnothorax longispinosus

    NASA Astrophysics Data System (ADS)

    Howard, Kenneth J.; Kennedy, David

    2007-11-01

    Mating behaviors of ants fall into two categories: female calling, in which a female alate releases pheromones that attract males, and male swarming, in which large male aggregations attract females. Female calling is common in species with queens that return to their natal nest to found colonies dependently after mating, while male swarming is common in species with queens that disperse to found independently. In some species that display both founding strategies, a queen-size polymorphism has evolved in which dependent-founding queens are smaller than independent-founding queens. Dependent founding is likely difficult if gynes (virgin queens) are mating in distant swarms. Therefore, a queen may adopt one or the other mating strategy based on its size and founding behavior. We investigated mating behaviors in the queen-polymorphic ant, Temnothorax longispinosus. Observations in laboratory mating arenas indicated that small gynes exhibited significantly lower flight activity than large gynes. Both forms mated in male swarms, and neither form exhibited female calling. The reduced flight activity of the small morph may facilitate returning to the natal nest after mating, provided the mating swarm is located nearby. Therefore, alternative colony-founding behaviors may be possible without the evolution of female-calling behavior; however, the reduced flight activity of small morphs may require that mating swarms are not distant from the natal nest.

  3. Alternative mating behaviors of the queen polymorphic ant Temnothorax longispinosus.

    PubMed

    Howard, Kenneth J; Kennedy, David

    2007-11-01

    Mating behaviors of ants fall into two categories: female calling, in which a female alate releases pheromones that attract males, and male swarming, in which large male aggregations attract females. Female calling is common in species with queens that return to their natal nest to found colonies dependently after mating, while male swarming is common in species with queens that disperse to found independently. In some species that display both founding strategies, a queen-size polymorphism has evolved in which dependent-founding queens are smaller than independent-founding queens. Dependent founding is likely difficult if gynes (virgin queens) are mating in distant swarms. Therefore, a queen may adopt one or the other mating strategy based on its size and founding behavior. We investigated mating behaviors in the queen-polymorphic ant, Temnothorax longispinosus. Observations in laboratory mating arenas indicated that small gynes exhibited significantly lower flight activity than large gynes. Both forms mated in male swarms, and neither form exhibited female calling. The reduced flight activity of the small morph may facilitate returning to the natal nest after mating, provided the mating swarm is located nearby. Therefore, alternative colony-founding behaviors may be possible without the evolution of female-calling behavior; however, the reduced flight activity of small morphs may require that mating swarms are not distant from the natal nest. PMID:17653686

  4. In vitro transmission and propagation of the bovine leukemia virus in monolayer cell cultures.

    PubMed

    Graves, D C; Ferrer, J F

    1976-11-01

    This study demonstrates that the bovine leukemia virus (BLV) can infect in vitro cells of human, simian, bovine, canine, caprine, ovine, and bat origin. Cultures of these cells, cocultivated with BLV-infected cells or inoculated with cell-free BLV preparations, continuoously showed the presence of cells with the internal BLV antigen as well as BLV-induced syncytia. Virus replication was abundant and increased with passage in bat lung cells and was moderate but constant in fetal canine thymus cells. The amounts of virus released by the simian DBS-FRhL-1 and caprine S-743 cultures were low to moderate during the first 4 to 8 weeks and decreased thereafter. In the infected fetal lamb spleen cell cultures, virus production was low and declined further with passage. Bovine embryonic spleen and human diploid embryonic lung WI-38 cell cultures produced very small amounts of virus only during the first two passages after inoculation despite the fact that they remained infected, as determined by the continuous presence of cell BLV antigen and syncytia. Morphologically and antigenically, the virus particles released by the monolayer cell cultures were indistinguishable from those found in short-term and long-term cultures of BLV-infected bovine lymphoid cells. Repeated electron microscopic examinations and serological tests showed that all the BLV-infected cultures, including those from which the infecting inocula were obtained, were free of the foamy-like bovine syncytial virus, parainfluenza 3 virus, infectious bovine rhinotracheitis virus, bovine viral diarrhea virus, and the maedi-like bovine R-29 virus. PMID:61801

  5. Replication of influenza A virus in swine umbilical cord epithelial stem-like cells

    PubMed Central

    Khatri, Mahesh; Chattha, Kuldeep S

    2015-01-01

    In this study, we describe the isolation and characterization of epithelial stem-like cells from the swine umbilical cord and their susceptibility to influenza virus infection. Swine umbilical cord epithelial stem cells (SUCECs) expressed stem cell and pluripotency associated markers such as SSEA-1, SSEA-4, TRA 1–60 and TRA 1–81 and Oct4. Morphologically, cells displayed polygonal morphology and were found to express epithelial markers; pancytokeratin, cytokeratin-18 and occludin; mesenchymal cell markers CD44, CD90 and haematopoietic cell marker CD45 were not detected on these cells. The cells had extensive proliferation and self- renewal properties. The cells also possessed immunomodulatory activity and inhibited the proliferation of T cells. Also, higher levels of anti-inflammatory cytokine IL-10 were detected in SUCEC-T cell co-cultures. The cells were multipotent and differentiated into lung epithelial cells when cultured in epithelial differentiation media. We also examined if SUCECs are susceptible to infection with influenza virus. SUCECs expressed sialic acid receptors, used by influenza virus for binding to cells. The 2009 pandemic influenza virus and swine influenza virus replicated in these cells. SUCECs due to their differentiation and immunoregulatory properties will be useful as cellular therapy in a pig model for human diseases. Additionally, our data indicate that influenza virus can infect SUCECs and may transmit influenza virus from mother to fetus through umbilical cord and transplantation of influenza virus-infected stem cells may transmit infection to recipients. Therefore, we propose that umbilical cord cells, in addition to other agents, should also be tested for influenza virus before cryopreservation for future use as a cell therapy for disease conditions. PMID:25517546

  6. FBJ osteosarcoma virus in tissue culture. III. Isolation and characterization of non-virus-producing FBJ-transformed cells.

    PubMed Central

    Levy, J A; Kazan, P L; Reilly, C A; Finkel, M P

    1978-01-01

    Hamster and rat cell lines have been established that have been transformed by FBJ murine sarcoma virus (FBJ-MuSV) but that do not produce virus. The hamster cell line originated from an osteosarcoma that appeared in a hamster inoculated at birth with an extract of a CFNo1 mouse FBJ-osteosarcoma. The rat cell line was obtained by transferring the FBJ-MuSV genome to normal rat kidney cells in the absence of the FBJ type C virus (FBJ-MuLV), which, usually in high concentration, accompanies the FBJ-MuSV. Both transformed hamster and rat cell lines contain the FBJ-MuSV genome, which can be rescued by ecotropic and xenotropic murine type C viruses. This rescued genome produces characteristic FBJ-MuSV foci in tissue culture and, in appropriate animal hosts, induces osteosarcomas typical of those induced by FBJ-MuSV. FBJ-MuSV was isolated originally from a parosteal osteosarcoma that occurred naturally in a mouse. Since there was no previous history of passage of the agent through any other animal species, these non-virus-producing hamster and rat cells transformed by FBJ-MuSV should be very helpful in molecular studies examining the origin of spontaneous sarcoma genomes in mice. PMID:206718

  7. Modelling the Impact of Cell-To-Cell Transmission in Hepatitis B Virus

    PubMed Central

    2016-01-01

    Cell-free virus is a well-recognized and efficient mechanism for the spread of hepatitis B virus (HBV) infection in the liver. Cell-to-cell transmission (CCT) can be a more efficient means of virus propagation. Despite experimental evidence implying CCT occurs in HBV, its relative impact is uncertain. We develop a 3-D agent-based model where each hepatocyte changes its viral state according to a dynamical process driven by cell-free virus infection, CCT and intracellular replication. We determine the relative importance of CCT in the development and resolution of acute HBV infection in the presence of cytolytic (CTL) and non-CTL mechanisms. T cell clearance number is defined as the minimum number of infected cells needed to be killed by each T cell at peak infection that results in infection clearance within 12 weeks with hepatocyte turnover (HT, number of equivalent livers) ≤3. We find that CCT has very little impact on the establishment of infection as the mean cccDNA copies/cell remains between 15 to 20 at the peak of the infection regardless of CCT strength. In contrast, CCT inhibit immune-mediated clearance of acute HBV infection as higher CCT strength requires higher T cell clearance number and increases the probability of T cell exhaustion. An effective non-CTL inhibition can counter these negative effects of higher strengths of CCT by supporting rapid, efficient viral clearance and with little liver destruction. This is evident as the T cell clearance number drops by approximately 50% when non-CTL inhibition is increased from 10% to 80%. Higher CCT strength also increases the probability of the incidence of fulminant hepatitis with this phenomenon being unlikely to arise for no CCT. In conclusion, we report the possibility of CCT impacting HBV clearance and its contribution to fulminant hepatitis. PMID:27560827

  8. Modelling the Impact of Cell-To-Cell Transmission in Hepatitis B Virus.

    PubMed

    Goyal, Ashish; Murray, John M

    2016-01-01

    Cell-free virus is a well-recognized and efficient mechanism for the spread of hepatitis B virus (HBV) infection in the liver. Cell-to-cell transmission (CCT) can be a more efficient means of virus propagation. Despite experimental evidence implying CCT occurs in HBV, its relative impact is uncertain. We develop a 3-D agent-based model where each hepatocyte changes its viral state according to a dynamical process driven by cell-free virus infection, CCT and intracellular replication. We determine the relative importance of CCT in the development and resolution of acute HBV infection in the presence of cytolytic (CTL) and non-CTL mechanisms. T cell clearance number is defined as the minimum number of infected cells needed to be killed by each T cell at peak infection that results in infection clearance within 12 weeks with hepatocyte turnover (HT, number of equivalent livers) ≤3. We find that CCT has very little impact on the establishment of infection as the mean cccDNA copies/cell remains between 15 to 20 at the peak of the infection regardless of CCT strength. In contrast, CCT inhibit immune-mediated clearance of acute HBV infection as higher CCT strength requires higher T cell clearance number and increases the probability of T cell exhaustion. An effective non-CTL inhibition can counter these negative effects of higher strengths of CCT by supporting rapid, efficient viral clearance and with little liver destruction. This is evident as the T cell clearance number drops by approximately 50% when non-CTL inhibition is increased from 10% to 80%. Higher CCT strength also increases the probability of the incidence of fulminant hepatitis with this phenomenon being unlikely to arise for no CCT. In conclusion, we report the possibility of CCT impacting HBV clearance and its contribution to fulminant hepatitis. PMID:27560827

  9. Intraspecific queen parasitism in a highly eusocial bee

    PubMed Central

    Wenseleers, Tom; Alves, Denise A.; Francoy, Tiago M.; Billen, Johan; Imperatriz-Fonseca, Vera L.

    2011-01-01

    Insect societies are well-known for their advanced cooperation, but their colonies are also vulnerable to reproductive parasitism. Here, we present a novel example of an intraspecific social parasitism in a highly eusocial bee, the stingless bee Melipona scutellaris. In particular, we provide genetic evidence which shows that, upon loss of the mother queen, many colonies are invaded by unrelated queens that fly in from unrelated hives nearby. The reasons for the occurrence of this surprising form of social parasitism may be linked to the fact that unlike honeybees, Melipona bees produce new queens in great excess of colony needs, and that this exerts much greater selection on queens to seek alternative reproductive options, such as by taking over other nests. Overall, our results are the first to demonstrate that queens in highly eusocial bees can found colonies not only via supersedure or swarming, but also by infiltrating and taking over other unrelated nests. PMID:20961883

  10. Hypomethylation of host cell DNA synthesized after infection or transformation of cells by herpes simplex virus

    SciTech Connect

    Macnab, J.C.M.; Adams, R.L.P.; Rinaldi, A.; Orr, A.; Clark, L.

    1988-04-01

    Infection of rat embryo cells with herpes simplex virus type 2 caused undermethylation of host cell DNA synthesized during infection. DNA made prior to infection was not demethylated, but some of its degradation products, including methyl dCMP, were incorporated into viral DNA. The use of mutant virus showed that some viral DNA synthesis appears to be required for the inhibition of methylation. Inhibition of methylation cannot be explained by an absence of DNA methyltransferase as the activity of this enzyme did not change during the early period of infection. Inhibition of host cell DNA methylation may be an important step in the transformation of cells by herpesviruses, and various transformed cell lines tested showed reduced levels of DNA methylation.

  11. Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion ▿

    PubMed Central

    Sharma, Nishi R.; Mani, Prashant; Nandwani, Neha; Mishra, Rajakishore; Rana, Ajay; Sarkar, Debi P.

    2010-01-01

    Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy. PMID:20164223

  12. Detection of C-type virus by immunoferritin technique in bat lung cell line chronically infected with bovine leucosis virus.

    PubMed

    Mihailescu, D; Patrascu, I V; Apostol, I; Mazilu, M

    1980-01-01

    Reported in this paper are morphological studies and tests for the detection of Type-C particles from a line of bat lung cells chronically infected with bovine leucosis virus. The immunoferritin technique was used. Ferritin labelling of Type-C particles was regularly accompanied by black-spot arrangement of ferritin around the virus envelope, which provided evidence to the specificity of this immunochemical technique. PMID:6260052

  13. Cell-cell contact promotes Ebola virus GP-mediated infection.

    PubMed

    Miao, Chunhui; Li, Minghua; Zheng, Yi-Min; Cohen, Fredric S; Liu, Shan-Lu

    2016-01-15

    Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Here we provide evidence that cell-cell contact promotes infection mediated by the glycoprotein (GP) of EBOV. Interestingly, expression of EBOV GP alone, even in the absence of retroviral Gag-Pol, is sufficient to transfer a retroviral vector encoding Tet-off from cell to cell. Cell-to-cell infection mediated by EBOV GP is blocked by inhibitors of actin polymerization, but appears to be less sensitive to KZ52 neutralization. Treatment of co-cultured cells with cathepsin B/L inhibitors, or an entry inhibitor 3.47 that targets the receptor NPC1 for virus binding, also blocks cell-to-cell infection. Cell-cell contact also enhances spread of rVSV bearing GP in monocytes and macrophages, the primary targets of natural EBOV infection. Altogether, our study reveals that cell-cell contact promotes EBOV GP-mediated infection, and provides new insight into understanding EBOV spread and viral pathogenesis. PMID:26655238

  14. Cell surface markers on epithelial-Burkitt hybrid cells superinfected with Epstein-Barr virus.

    PubMed

    Glaser, R; Lenoir, G; Ferrone, S; Pellegrino, M A; de-Thé, G

    1977-07-01

    Attempts were made to superinfect two epithelial-Burkitt hybrid cell lines, designated D98/HR-1 and D98/Raji, with Epstein-Barr virus (EBV) and to investigate the expression of some cell surfacr markers including histocompatibility antigens, and the presence of B-cell markers, such as receptors for the third complement component and for monkey red blood cells. Successful superinfection of D98/HR-1 cells with EBV was made evident by the expression of early antigen and, to a lesser extent, virus capsid antigen. Only a rare D98/Raji cell was found to be positive for early antigen. The histocompatibility antigens of the parental cell lines D98, HR-1, and Raji were expressed on the surfaces of the hybrid cells. Receptors for third complement components b and d were not detected on the hybrid cells or on the D98P OR HR-1 cell lines; they were found, however, on the Raji cells, indicating that EBV receptors and complement receptors can be separated. The significance of the infection of the hybrid cells with EBV and the expression of cell surface markers is described. PMID:193641

  15. Canine Distemper Virus Epithelial Cell Infection Is Required for Clinical Disease but Not for Immunosuppression

    PubMed Central

    Sawatsky, Bevan; Wong, Xiao-Xiang; Hinkelmann, Sarah; Cattaneo, Roberto

    2012-01-01

    To characterize the importance of infection of epithelial cells for morbillivirus pathogenesis, we took advantage of the severe disease caused by canine distemper virus (CDV) in ferrets. To obtain a CDV that was unable to enter epithelial cells but retained the ability to enter immune cells, we transferred to its attachment (H) protein two mutations shown to interfere with the interaction of measles virus H with its epithelial receptor, human nectin-4. As expected for an epithelial receptor (EpR)-blind CDV, this virus infected dog and ferret epithelial cells inefficiently and did not cause cell fusion or syncytium formation. On the other hand, the EpR-blind CDV replicated in cells expressing canine signaling lymphocyte activation molecule (SLAM), the morbillivirus immune cell receptor, with similar kinetics to those of wild-type CDV. While ferrets infected with wild-type CDV died within 12 days after infection, after developing severe rash and fever, animals infected with the EpR-blind virus showed no clinical signs of disease. Nevertheless, both viruses spread rapidly and efficiently in immune cells, causing similar levels of leukopenia and inhibition of lymphocyte proliferation activity, two indicators of morbillivirus immunosuppression. Infection was documented for airway epithelia of ferrets infected with wild-type CDV but not for those of animals infected with the EpR-blind virus, and only animals infected with wild-type CDV shed virus. Thus, epithelial cell infection is necessary for clinical disease and efficient virus shedding but not for immunosuppression. PMID:22278252

  16. Effect of caffeine on induction of endogenous type C virus in mouse cells in vitro

    SciTech Connect

    Niwa, O.; Sugahara, T.

    1981-08-01

    The effect of caffeine on the expression of murine endogenous virus in mouse cells induced by radiation and chemicals was studied. Postirradiation treatment of K-BALB cells with caffeine enhanced cell killing as well as the induction of xenotropic virus after ultraviolet light irradiation. The degree of enhancement for the virus induction was comparable to that for cell killing. On the other hand, colony-forming ability and the expression of xenotropic virus of K-BALB cells after X-irradiation were unaffected by caffeine. These data suggest a linear relationship between the degree of endogenous virus expression and the amount of lethal damages after irradiation. For induction by halogenated pyrimidines, a 24-hr incubation of AKR2B cells with caffeine after 5-iodo-2'-deoxyuridine treatment resulted in marked suppression of the expression of ecotropic virus. On the contrary, in K-BALB cells, caffeine exerted only a small effect on 5-iodo-2'-deoxyuridine-induced expression of ecotropic and xenotropic viruses. These results indicate that, although using the same inducing agent, the pathway of endogenous virus induction may be different for AKR2B cells and for K-BALB cells.

  17. Mechanisms of pathogenesis induced by bovine leukemia virus as a model for human T-cell leukemia virus

    PubMed Central

    Aida, Yoko; Murakami, Hironobu; Takahashi, Masahiko; Takeshima, Shin-Nosuke

    2013-01-01

    Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up a unique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia and a long latency period. These viruses encode at least two regulatory proteins, namely, Tax and Rex, in the pX region located between the env gene and the 3′ long terminal repeat. The Tax protein is a key contributor to the oncogenic potential of the virus, and is also the key protein involved in viral replication. However, BLV infection is not sufficient for leukemogenesis, and additional events such as gene mutations must take place. In this review, we first summarize the similarities between the two viruses in terms of genomic organization, virology, and pathology. We then describe the current knowledge of the BLV model, which may also be relevant for the understanding of leukemogenesis caused by HTLV-1. In addition, we address our improved understanding of Tax functions through the newly identified BLV Tax mutants, which have a substitution between amino acids 240 and 265. PMID:24265629

  18. Inhibition of Sindbis virus maturation after treatment of infected cells with trypsin.

    PubMed Central

    Adams, R H; Brown, D T

    1982-01-01

    Brief treatment of Sindbis virus-infected BHK-21 or Vero cells with low concentrations of trypsin irreversibly blocked further production of progeny virions after removal of the enzyme. The inhibitory effects of the trypsin treatment could only be demonstrated in cells in which virus infection was established; optimal inhibition occurred at ca. 3 h postinfection. Production of virus structural proteins PE2, E1, and C occurred at normal levels in inhibited cells. PE2 and E1 were also transported to the cell plasma membrane during inhibition; however, PE2 was not cleaved to E2, and little capsid protein became membrane associated relative to control cells. Although trypsin treatment had no effect on Sindbis protein synthesis, the production of both 26S and 42S RNA was greatly reduced. Similar trypsin treatment of BHK cells infected with vesicular stomatitis virus had no detectable effect on the course of virus infection. Images PMID:6281478

  19. T-cell memory: lessons from Epstein-Barr virus infection in man.

    PubMed Central

    Rickinson, A B; Callan, M F; Annels, N E

    2000-01-01

    Epstein-Barr virus offers an ideal opportunity to follow the human T-cell response to a virus infection over time from its acute primary phase, as seen in infectious mononucleosis patients, into the memory phase that accompanies life-long virus persistence. Here we review recent evidence on the development and maturation of cytotoxic T-cell memory using this viral system. PMID:10794060

  20. Hepatic Cells Derived from Induced Pluripotent Stem Cells of Pigtail Macaques Support Hepatitis C Virus infection

    PubMed Central

    Sourisseau, Marion; Goldman, Orit; He, Wenqian; Gori, Jennifer L.; Kiem, Hans-Peter; Gouon-Evans, Valerie; Evans, Matthew J.

    2013-01-01

    The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less-restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection. PMID:23891978

  1. Host cell kinases and the hepatitis C virus life cycle.

    PubMed

    Colpitts, Che C; Lupberger, Joachim; Doerig, Christian; Baumert, Thomas F

    2015-10-01

    Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases. PMID:25896387

  2. Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation.

    PubMed Central

    Boettiger, D; Durban, E

    1984-01-01

    The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus. PMID:6699939

  3. Hepatitis C virus - associated B cell non-Hodgkin's lymphoma.

    PubMed

    Mihăilă, Romeo-Gabriel

    2016-07-21

    The hepatitis C virus (HCV) infected patients are prone to develop bone marrow or various tissue infiltrates with monoclonal B cells, monoclonal B lymphocytosis or different types of B cell non-Hodgkin's lymphoma (BCNHL), of which the most common are splenic marginal zone BCNHL, diffuse large BCNHL and follicular lymphoma. The association between chronic HCV infection and non Hodgkin's lymphoma has been observed especially in areas with high prevalence of this viral infection. Outside the limitations of some studies that have been conducted, there are also geographic, environmental, and genetic factors that contribute to the epidemiological differences. Various microenvironmental signals, such as cytokines, viral antigenic external stimulation of lymphocyte receptors by HCV antigens, and intercellular interactions contribute to B cell proliferation. HCV lymphotropism and chronic antigenic stimulation are involved in B-lymphocyte expansion, as mixted cryoglobulinemia or monoclonal gammopathy of undetermined significance, which can progress to BCNHL. HCV replication in B lymphocytes has oncogenic effect mediated by intracellular HCV proteins. It is also involved in an important induction of reactive oxygen species that can lead to permanent B lymphocyte damage, as DNA mutations, after binding to surface B-cell receptors. Post-transplant lymphoproliferative disorder could appear and it has a multiclonal potentiality that may develop into different types of lymphomas. The hematopoietic stem cell transplant made for lymphoma in HCV-infected patients can increase the risk of earlier progression to liver fibrosis and cirrhosis. HCV infected patients with indolent BCNHL who receive antiviral therapy can be potentially cured. Viral clearance was related to lymphoma response, fact that highlights the probable involvement of HCV in lymphomagenesis. Direct acting antiviral drugs could be a solution for the patients who did not tolerate or respond to interferon, as they seem to

  4. Hepatitis C virus - associated B cell non-Hodgkin's lymphoma

    PubMed Central

    Mihăilă, Romeo-Gabriel

    2016-01-01

    The hepatitis C virus (HCV) infected patients are prone to develop bone marrow or various tissue infiltrates with monoclonal B cells, monoclonal B lymphocytosis or different types of B cell non-Hodgkin’s lymphoma (BCNHL), of which the most common are splenic marginal zone BCNHL, diffuse large BCNHL and follicular lymphoma. The association between chronic HCV infection and non Hodgkin’s lymphoma has been observed especially in areas with high prevalence of this viral infection. Outside the limitations of some studies that have been conducted, there are also geographic, environmental, and genetic factors that contribute to the epidemiological differences. Various microenvironmental signals, such as cytokines, viral antigenic external stimulation of lymphocyte receptors by HCV antigens, and intercellular interactions contribute to B cell proliferation. HCV lymphotropism and chronic antigenic stimulation are involved in B-lymphocyte expansion, as mixted cryoglobulinemia or monoclonal gammopathy of undetermined significance, which can progress to BCNHL. HCV replication in B lymphocytes has oncogenic effect mediated by intracellular HCV proteins. It is also involved in an important induction of reactive oxygen species that can lead to permanent B lymphocyte damage, as DNA mutations, after binding to surface B-cell receptors. Post-transplant lymphoproliferative disorder could appear and it has a multiclonal potentiality that may develop into different types of lymphomas. The hematopoietic stem cell transplant made for lymphoma in HCV-infected patients can increase the risk of earlier progression to liver fibrosis and cirrhosis. HCV infected patients with indolent BCNHL who receive antiviral therapy can be potentially cured. Viral clearance was related to lymphoma response, fact that highlights the probable involvement of HCV in lymphomagenesis. Direct acting antiviral drugs could be a solution for the patients who did not tolerate or respond to interferon, as they

  5. Influenza virus assays based on virus‐inducible reporter cell lines

    PubMed Central

    Li, Yunsheng; Larrimer, Audrey; Curtiss, Teresa; Kim, Jaekyung; Jones, Abby; Baird‐Tomlinson, Heather; Pekosz, Andrew; Olivo, Paul D.

    2009-01-01

    Background  Virus‐inducible reporter genes have been used as the basis of virus detection and quantitation assays for a number of viruses. A strategy for influenza A virus‐induction of a reporter gene was recently described. In this report, we describe the extension of this strategy to influenza B virus, the generation of stable cell lines with influenza A and B virus‐inducible reporter genes, and the use of these cells in various clinically relevant viral assays. Each of the cell lines described herein constitutively express an RNA transcript that contains a reporter gene coding region flanked by viral 5′‐ and 3′‐untranslated regions (UTR) and therefore mimics an influenza virus genomic segment. Upon infection of the cells with influenza virus the virus‐inducible reporter gene segment (VIRGS) is replicated and transcribed by the viral polymerase complex resulting in reporter gene expression. Findings  Reporter gene induction occurs after infection with a number of laboratory strains and clinical isolates of influenza virus including several H5N1 strains. The induction is dose‐dependent and highly specific for influenza A or influenza B viruses. Conclusions  These cell lines provide the basis of simple, rapid, and objective assays that involve virus quantitation such as determination of viral titer, assessment of antiviral susceptibility, and determination of antibody neutralization titer. These cell lines could be very useful for influenza virus researchers and vaccine manufacturers. PMID:21462401

  6. Effect of Host Cell on Distribution of a Lysosomal Enzyme During Virus Infection

    PubMed Central

    Sato, Kosaburo; Righthand, Fay; Karzon, David T.

    1971-01-01

    The time of appearance of a lysosomal enzyme, β-glucuronidase, in the medium of cells infected with either measles virus or echovirus 6 varied with the host cell system. Replication and release of virus preceded leakage of β-glucuronidase from green monkey kidney cells. In contrast, extracellular enzyme appeared before replication and release of virus in human amnion cells. Hydrocortisone depressed enzyme leakage but did not retard replication of measles virus or viral-induced cytopathology. The intracellular distribution of β-glucuronidase in uninfected and measles virus-infected cells was also studied. Measles virus infection altered the position of particulate-bound β-glucuronidase in linear sucrose gradients prior to substantial release of this enzyme intra- and extracellularly. At early stages in infection, most of the cell-associated virus banded with particulate-bound enzyme in the middle of the gradient. As infection progressed, separation of measles virus infectivity from enzyme activity occurred, and intracellular virus was recovered near the meniscus of sucrose gradients. PMID:5000115

  7. Dendritic Cells in Dengue Virus Infection: Targets of Virus Replication and Mediators of Immunity

    PubMed Central

    Schmid, Michael A.; Diamond, Michael S.; Harris, Eva

    2014-01-01

    Dendritic cells (DCs) are sentinels of the immune system and detect pathogens at sites of entry, such as the skin. In addition to the ability of DCs to control infections directly via their innate immune functions, DCs help to prime adaptive B- and T-cell responses by processing and presenting antigen in lymphoid tissues. Infected Aedes aegypti or Aedes albopictus mosquitoes transmit the four dengue virus (DENV) serotypes to humans while probing for small blood vessels in the skin. DENV causes the most prevalent arthropod-borne viral disease in humans, yet no vaccine or specific therapeutic is currently licensed. Although primary DENV infection confers life-long protective immunity against re-infection with the same DENV serotype, secondary infection with a different DENV serotype can lead to increased disease severity via cross-reactive T-cells or enhancing antibodies. This review summarizes recent findings in humans and animal models about DENV infection of DCs, monocytes, and macrophages. We discuss the dual role of DCs as both targets of DENV replication and mediators of innate and adaptive immunity, and summarize immune evasion strategies whereby DENV impairs the function of infected DCs. We suggest that DCs play a key role in priming DENV-specific neutralizing or potentially harmful memory B- and T-cell responses, and that future DC-directed therapies may help induce protective memory responses and reduce dengue pathogenesis. PMID:25566258

  8. Propagation of viruses infecting waterfowl on continuous cell lines of Muscovy duck (Cairina moschata) origin.

    PubMed

    Mészáros, István; Tóth, Renáta; Bálint, Adám; Dán, Adám; Jordan, Ingo; Zádori, Zoltán

    2014-01-01

    Duck circovirus, duck hepatitis A virus 1, goose parvovirus and goose haemorrhagic polyomavirus are economically damaging pathogens of waterfowl, and replicate poorly or not at all in established cell lines. AGE1.CR, AGE1.CR.pIX and AGE1.CS cell lines, originating from the Muscovy duck, were tested for their suitability to isolate and identify these viruses. Immunofluorescence (IF) and quantitative polymerase chain reaction investigations verified that all cell lines are permissive for all four viruses; however, AGE1.CR.pIX proved to be the most productive and most sensitive for viral infection. IF experiments revealed that the time of one infectious cycle is approximately 12 to 14 h in the AGE1.CR.pIX cells in the case of the three DNA viruses, while it is 10 to 12 h for DHAV-1. Specific viral infectivity and the limit of detection by IF varied between 55 and 1484 copies, depending on the viruses and cell lines. Despite the high sensitivity of the cell lines for viruses, their viral productivity remained relatively low for the investigated field isolates. However, optimization of virus infection and/or the adaptation of the viruses to the cells can raise viral productivity and can make these cell lines suitable for vaccine development and production. PMID:24992264

  9. In Vitro Host Range of the Hz-1 Non-Occluded Virus in Arthropod Cell Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A total of 13 insect cell lines spanning 4 Orders and including Lepidoptera, Coleoptera, Diptera and Homoptera were tested for their ability to replicate the non-occluded virus Hz-1. Only the Lepidpteran cell lines were able to support replication of the virus with TN-CL1 and BCIRL-HZ-AM1 producing ...

  10. Genome Sequence of the Parainfluenza Virus 5 Strain That Persistently Infects AGS Cells

    PubMed Central

    Wignall-Fleming, Elizabeth; Young, Dan F.; Goodbourn, Steve; Davison, Andrew J.

    2016-01-01

    We have sequenced the parainfluenza virus 5 strain that persistently infects the commonly used AGS human cell line without causing cytopathology. This virus is most closely related to human strains, indicating that it may have originated from biopsy material or from laboratory contamination during generation of the cell line. PMID:27445371

  11. A primary chicken tracheal cell culture system for the study of infection with avian respiratory viruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the airway epithelium. Here we describe the development and optimization of conditions for culture of tracheal epithelial cells from chicken embryos as well as their use in st...

  12. Continuous Influenza Virus Production in Cell Culture Shows a Periodic Accumulation of Defective Interfering Particles

    PubMed Central

    Pflugmacher, Antje; Behrendt, Ilona; Jordan, Ingo; Flockerzi, Dietrich; Genzel, Yvonne; Reichl, Udo

    2013-01-01

    Influenza viruses are a major public health burden during seasonal epidemics and a continuous threat due to their potential to cause pandemics. Annual vaccination provides the best protection against the contagious respiratory illness caused by influenza viruses. However, the current production capacities for influenza vaccines are insufficient to meet the increasing demands. We explored the possibility to establish a continuous production process for influenza viruses using the duck-derived suspension cell line AGE1.CR. A two-stage bioreactor setup was designed in which cells were cultivated in a first stirred tank reactor where an almost constant cell concentration was maintained. Cells were then constantly fed to a second bioreactor where virus infection and replication took place. Using this two-stage reactor system, it was possible to continuously produce influenza viruses. Surprisingly, virus titers showed a periodic increase and decrease during the run-time of 17 days. These titer fluctuations were caused by the presence of defective interfering particles (DIPs), which we detected by PCR. Mathematical modeling confirmed this observation showing that constant virus titers can only emerge in the absence of DIPs. Even with very low amounts of DIPs in the seed virus and very low rates for de novo DIP generation, defective viruses rapidly accumulate and, therefore, represent a serious challenge for continuous vaccine production. Yet, the continuous replication of influenza virus using a two-stage bioreactor setup is a novel tool to study aspects of viral evolution and the impact of DIPs. PMID:24039749

  13. Androgen-independent proliferation of LNCaP prostate cancer cells infected by xenotropic murine leukemia virus-related virus

    SciTech Connect

    Kakoki, Katsura; Kamiyama, Haruka; Izumida, Mai; Yashima, Yuka; Hayashi, Hideki; Yamamoto, Naoki; Matsuyama, Toshifumi; Igawa, Tsukasa; Sakai, Hideki; Kubo, Yoshinao

    2014-04-25

    Highlights: • XMRV infection induces androgen-independent growth in LNCaP cells. • XMRV infection reduces expression of androgen receptor. • XMRV promotes appearance of androgen blocker-resistant prostate cancer cells. - Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a novel gammaretrovirus that was originally isolated from human prostate cancer. It is now believed that XMRV is not the etiologic agent of prostate cancer. An analysis of murine leukemia virus (MLV) infection in various human cell lines revealed that prostate cancer cell lines are preferentially infected by XMRV, and this suggested that XMRV infection may confer some sort of growth advantage to prostate cancer cell lines. To examine this hypothesis, androgen-dependent LNCaP cells were infected with XMRV and tested for changes in certain cell growth properties. We found that XMRV-infected LNCaP cells can proliferate in the absence of the androgen dihydrotestosterone. Moreover, androgen receptor expression is significantly reduced in XMRV-infected LNCaP cells. Such alterations were not observed in uninfected and amphotropic MLV-infected LNCaP cells. This finding explains why prostate cancer cell lines are preferentially infected with XMRV.

  14. Expression of the infectious salmon anemia virus receptor on atlantic salmon endothelial cells correlates with the cell tropism of the virus.

    PubMed

    Aamelfot, Maria; Dale, Ole Bendik; Weli, Simon Chioma; Koppang, Erling Olaf; Falk, Knut

    2012-10-01

    Infectious salmon anemia (ISA) is a World Organization for Animal Health (OIE)-listed disease of farmed Atlantic salmon, characterized by slowly developing anemia and circulatory disturbances. The disease is caused by ISA virus (ISAV) in the Orthomyxoviridae family; hence, it is related to influenza. Here we explore the pathogenesis of ISA by focusing on virus tropism, receptor tissue distribution, and pathological changes in experimentally and naturally infected Atlantic salmon. Using immunohistochemistry on ISAV-infected Atlantic salmon tissues with antibody to viral nucleoprotein, endotheliotropism was demonstrated. Endothelial cells lining the circulatory system were found to be infected, seemingly noncytolytic, and without vasculitis. No virus could be found in necrotic parenchymal cells. From endothelium, the virus budded apically and adsorbed to red blood cells (RBCs). No infection or replication within RBCs was detected, but hemophagocytosis was observed, possibly contributing to the severe anemia in fish with this disease. Similarly to what has been done in studies of influenza, we examined the pattern of virus attachment by using ISAV as a probe. Here we detected the preferred receptor of ISAV, 4-O-acetylated sialic acid (Neu4,5Ac(2)). To our knowledge, this is the first report demonstrating the in situ distribution of this sialic acid derivate. The pattern of virus attachment mirrored closely the distribution of infection, showing that the virus receptor is important for cell tropism, as well as for adsorption to RBCs. PMID:22811536

  15. Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

    PubMed Central

    Ink, B S; Gilbert, C S; Evan, G I

    1995-01-01

    The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family. PMID:7815529

  16. Fluorescent Protein-Tagged Sindbis Virus E2 Glycoprotein Allows Single Particle Analysis of Virus Budding from Live Cells

    PubMed Central

    Jose, Joyce; Tang, Jinghua; Taylor, Aaron B.; Baker, Timothy S.; Kuhn, Richard J.

    2015-01-01

    Sindbis virus (SINV) is an enveloped, mosquito-borne alphavirus. Here we generated and characterized a fluorescent protein-tagged (FP-tagged) SINV and found that the presence of the FP-tag (mCherry) affected glycoprotein transport to the plasma membrane whereas the specific infectivity of the virus was not affected. We examined the virions by transmission electron cryo-microscopy and determined the arrangement of the FP-tag on the surface of the virion. The fluorescent proteins are arranged icosahedrally on the virus surface in a stable manner that did not adversely affect receptor binding or fusion functions of E2 and E1, respectively. The delay in surface expression of the viral glycoproteins, as demonstrated by flow cytometry analysis, contributed to a 10-fold reduction in mCherry-E2 virus titer. There is a 1:1 ratio of mCherry to E2 incorporated into the virion, which leads to a strong fluorescence signal and thus facilitates single-particle tracking experiments. We used the FP-tagged virus for high-resolution live-cell imaging to study the spatial and temporal aspects of alphavirus assembly and budding from mammalian cells. These processes were further analyzed by thin section microscopy. The results demonstrate that SINV buds from the plasma membrane of infected cells and is dispersed into the surrounding media or spread to neighboring cells facilitated by its close association with filopodial extensions. PMID:26633461

  17. The V domain of dog PVRL4 (nectin-4) mediates canine distemper virus entry and virus cell-to-cell spread

    SciTech Connect

    Delpeut, Sebastien; Noyce, Ryan S.; Richardson, Christopher D.

    2014-04-15

    The entry of canine distemper virus (CDV) is a multistep process that involves the attachment of CDV hemagglutinin (H) to its cellular receptor, followed by fusion between virus and cell membranes. Our laboratory recently identified PVRL4 (nectin-4) to be the epithelial receptor for measles and canine distemper viruses. In this study, we demonstrate that the V domain of PVRL4 is critical for CDV entry and virus cell-to-cell spread. Furthermore, four key amino acid residues within the V domain of dog PVRL4 and two within the CDV hemagglutinin were shown to be essential for receptor-mediated virus entry. - Highlights: • PVRL4 (nectin-4) is the epithelial cell receptor for measles and canine distemper viruses. • V domain of PVRL4 is critical for CDV entry, cell-to-cell spread, and syncytia formation. • Chimeric PVRL1 backbone substituted with the V domain of PVRL4 can function as a receptor. • Amino acids (F132/P133/A134/G135) within the V domain are essential for PVRL4 receptor activity. • Amino acids (P493/Y539) within CDV H protein are essential for PVRL4 receptor interaction.

  18. Antimicrobial Peptides from Amphibian Skin Potently Inhibit Human Immunodeficiency Virus Infection and Transfer of Virus from Dendritic Cells to T Cells

    PubMed Central

    VanCompernolle, Scott E.; Taylor, R. Jeffery; Oswald-Richter, Kyra; Jiang, Jiyang; Youree, Bryan E.; Bowie, John H.; Tyler, Michael J.; Conlon, J. Michael; Wade, David; Aiken, Christopher; Dermody, Terence S.; KewalRamani, Vineet N.; Rollins-Smith, Louise A.; Unutmaz, Derya

    2005-01-01

    Topical antimicrobicides hold great promise in reducing human immunodeficiency virus (HIV) transmission. Amphibian skin provides a rich source of broad-spectrum antimicrobial peptides including some that have antiviral activity. We tested 14 peptides derived from diverse amphibian species for the capacity to inhibit HIV infection. Three peptides (caerin 1.1, caerin 1.9, and maculatin 1.1) completely inhibited HIV infection of T cells within minutes of exposure to virus at concentrations that were not toxic to target cells. These peptides also suppressed infection by murine leukemia virus but not by reovirus, a structurally unrelated nonenveloped virus. Preincubation with peptides prevented viral fusion to target cells and disrupted the HIV envelope. Remarkably, these amphibian peptides also were highly effective in inhibiting the transfer of HIV by dendritic cells (DCs) to T cells, even when DCs were transiently exposed to peptides 8 h after virus capture. These data suggest that amphibian-derived peptides can access DC-sequestered HIV and destroy the virus before it can be transferred to T cells. Thus, amphibian-derived antimicrobial peptides show promise as topical inhibitors of mucosal HIV transmission and provide novel tools to understand the complex biology of HIV capture by DCs. PMID:16140737

  19. Herpes simplex virus infects most cell types in vitro: clues to its success

    PubMed Central

    2011-01-01

    Herpes simplex virus (HSV) type-1 and type-2 have evolved numerous strategies to infect a wide range of hosts and cell types. The result is a very successful prevalence of the virus in the human population infecting 40-80% of people worldwide. HSV entry into host cell is a multistep process that involves the interaction of the viral glycoproteins with various cell surface receptors. Based on the cell type, HSV enter into host cell using different modes of entry. The combination of various receptors and entry modes has resulted in a virus that is capable of infecting virtually all cell types. Identifying the common rate limiting steps of the infection may help the development of antiviral agents that are capable of preventing the virus entry into host cell. In this review we describe the major features of HSV entry that have contributed to the wide susceptibility of cells to HSV infection. PMID:22029482

  20. Inhibition of apoptosis in human immunodeficiency virus-infected cells enhances virus production and facilitates persistent infection.

    PubMed Central

    Antoni, B A; Sabbatini, P; Rabson, A B; White, E

    1995-01-01

    Apoptosis is one of several mechanisms by which human immunodeficiency virus type 1 (HIV-1) exerts its cytopathic effects. CD4+ Jurkat T-cell lines overexpressing the adenovirus E1B 19K protein, a potent inhibitor of apoptosis, were used to examine the consequences of inhibition of apoptosis during acute and chronic HIV-1 infections. E1B 19K protein expression inhibited HIV-induced apoptosis, enhanced virus production, and established high levels of persistent viral infection. One E1B 19K-expressing line appeared to undergo HIV-induced death via a nonapoptotic mechanism, illustrating that HIV infection results in lymphocyte depletion through multiple pathways. Increased virus production associated with sustained cell viability suggests that therapeutic approaches involving inhibition of HIV-induced programmed cell death may be problematic. PMID:7884884

  1. Visualizing interactions between Sindbis virus and cells by single particle tracking

    NASA Astrophysics Data System (ADS)

    Williard, Mary

    2005-03-01

    Sindbis virus infects both mammalian and insect cells. Though not pathogenic in humans, Sindbis is a model for many mosquito- borne viruses that cause human disease, such as West Nile virus. We have used real-time single particle fluorescence microscopy to observe individual Sindbis virus particles as they infect living cells. Fluorescent labels were incorporated into both the viral coat proteins and the lipid envelope of the virus. Kinetics characteristic of free diffusion in solution, slower diffusion inside cells, attachment to spots on the cell surface, and motor protein transport inside cells have been observed. Dequenching of the membrane label is used to report membrane fusion events during the infection process. Tracking individual viral particles allows multiple pathways to be determined without the requirement of synchronicity.

  2. Subcellular distribution of swine vesicular disease virus proteins and alterations induced in infected cells: A comparative study with foot-and-mouth disease virus and vesicular stomatitis virus

    SciTech Connect

    Martin-Acebes, Miguel A.; Gonzalez-Magaldi, Monica; Rosas, Maria F.; Borrego, Belen; Brocchi, Emiliana; Armas-Portela, Rosario; Sobrino, Francisco

    2008-05-10

    The intracellular distribution of swine vesicular disease virus (SVDV) proteins and the induced reorganization of endomembranes in IBRS-2 cells were analyzed. Fluorescence to new SVDV capsids appeared first upon infection, concentrated in perinuclear circular structures and colocalized to dsRNA. As in foot-and-mouth disease virus (FMDV)-infected cells, a vesicular pattern was predominantly found in later stages of SVDV capsid morphogenesis that colocalized with those of non-structural proteins 2C, 2BC and 3A. These results suggest that assembly of capsid proteins is associated to the replication complex. Confocal microscopy showed a decreased fluorescence to ER markers (calreticulin and protein disulfide isomerase), and disorganization of cis-Golgi gp74 and trans-Golgi caveolin-1 markers in SVDV- and FMDV-, but not in vesicular stomatitis virus (VSV)-infected cells. Electron microscopy of SVDV-infected cells at an early stage of infection revealed fragmented ER cisternae with expanded lumen and accumulation of large Golgi vesicles, suggesting alterations of vesicle traffic through Golgi compartments. At this early stage, FMDV induced different patterns of ER fragmentation and Golgi alterations. At later stages of SVDV cytopathology, cells showed a completely vacuolated cytoplasm containing vesicles of different sizes. Cell treatment with brefeldin A, which disrupts the Golgi complex, reduced SVDV ({approx} 5 log) and VSV ({approx} 4 log) titers, but did not affect FMDV growth. Thus, three viruses, which share target tissues and clinical signs in natural hosts, induce different intracellular effects in cultured cells.

  3. Proteomic Analysis of Chikungunya Virus Infected Microgial Cells

    PubMed Central

    Abere, Bizunesh; Wikan, Nitwara; Ubol, Sukathida; Auewarakul, Prasert; Paemanee, Atchara; Kittisenachai, Suthathip; Roytrakul, Sittiruk; Smith, Duncan R.

    2012-01-01

    Chikungunya virus (CHIKV) is a recently re-emerged public health problem in many countries bordering the Indian Ocean and elsewhere. Chikungunya fever is a relatively self limiting febrile disease, but the consequences of chikungunya fever can include a long lasting, debilitating arthralgia, and occasional neurological involvement has been reported. Macrophages have been implicated as an important cell target of CHIKV with regards to both their role as an immune mediator, as well evidence pointing to long term viral persistence in these cells. Microglial cells are the resident brain macrophages, and so this study sought to define the proteomic changes in a human microglial cell line (CHME-5) in response to CHIKV infection. GeLC-MS/MS analysis of CHIKV infected and mock infected cells identified some 1455 individual proteins, of which 90 proteins, belonging to diverse cellular pathways, were significantly down regulated at a significance level of p<0.01. Analysis of the protein profile in response to infection did not support a global inhibition of either normal or IRES-mediated translation, but was consistent with the targeting of specific cellular pathways including those regulating innate antiviral mechanisms. PMID:22514668

  4. Nano-indentation experiments: from viruses to cells

    NASA Astrophysics Data System (ADS)

    Roos, Wouter; Vorselen, Daan; van Loon, Jack; Wuite, Gijs

    2012-02-01

    Over the last years AFM imaging and nanoindentation have become an indispensable tool for biophysical studies in liquid at the nano- and micro-scale. We look at both these length scales, at the cellular as well as the sub-cellular level. In particular, we perform combined imaging and force spectroscopy experiments on viral particles to elucidate their structure and mechanics [1]. These studies revealed that Noro virus has found an intriguing way to increase its mechanical strength. These self-assembling, natural nanoparticles incorporate a pre-stress during assembly, consolidating the structure of its protein shell that protects the genome [2]. Next, we studied whole cells. Mechanical loading is increasingly recognized as an important stimulus to cells. Establishing the local viscoelastic properties within a cell is vital to the understanding of the underlying mechanisms of cytoskeletal changes in response to these stimuli. We study the mechanical response of mammalian bone forming (osteoblast-like) cells on a substrate of physiological stiffness using spherical, μm-sized probes and we compare these results to the properties of bone forming cells originating from fish (teleosts).[4pt] [1] Roos et al. Nature Physics (2010), 6:733[0pt] [2] Baclayon et al. Nano Letters (2011), 11:4865

  5. Infectio: a Generic Framework for Computational Simulation of Virus Transmission between Cells

    PubMed Central

    Yakimovich, Artur; Yakimovich, Yauhen; Schmid, Michael; Mercer, Jason; Sbalzarini, Ivo F.

    2016-01-01

    ABSTRACT Viruses spread between cells, tissues, and organisms by cell-free and cell-cell mechanisms, depending on the cell type, the nature of the virus, or the phase of the infection cycle. The mode of viral transmission has a large impact on disease development, the outcome of antiviral therapies or the efficacy of gene therapy protocols. The transmission mode of viruses can be addressed in tissue culture systems using live-cell imaging. Yet even in relatively simple cell cultures, the mechanisms of viral transmission are difficult to distinguish. Here we present a cross-platform software framework called “Infectio,” which is capable of simulating transmission phenotypes in tissue culture of virtually any virus. Infectio can estimate interdependent biological parameters, for example for vaccinia virus infection, and differentiate between cell-cell and cell-free virus spreading. Infectio assists in elucidating virus transmission mechanisms, a feature useful for designing strategies of perturbing or enhancing viral transmission. The complexity of the Infectio software is low compared to that of other software commonly used to quantitate features of cell biological images, which yields stable and relatively error-free output from Infectio. The software is open source (GPLv3 license), and operates on the major platforms (Windows, Mac, and Linux). The complete source code can be downloaded from http://infectio.github.io/index.html. IMPORTANCE Infectio presents a generalized platform to analyze virus infection spread between cells. It allows the simulation of plaque phenotypes from image-based assays. Viral plaques are the result of virus spreading from primary infected cells to neighboring cells. This is a complex process and involves neighborhood effects at cell-cell contact sites or fluid dynamics in the extracellular medium. Infectio differentiates between two major modes of virus transmission between cells, allowing in silico testing of hypotheses about

  6. Infectio: a Generic Framework for Computational Simulation of Virus Transmission between Cells.

    PubMed

    Yakimovich, Artur; Yakimovich, Yauhen; Schmid, Michael; Mercer, Jason; Sbalzarini, Ivo F; Greber, Urs F

    2016-01-01

    Viruses spread between cells, tissues, and organisms by cell-free and cell-cell mechanisms, depending on the cell type, the nature of the virus, or the phase of the infection cycle. The mode of viral transmission has a large impact on disease development, the outcome of antiviral therapies or the efficacy of gene therapy protocols. The transmission mode of viruses can be addressed in tissue culture systems using live-cell imaging. Yet even in relatively simple cell cultures, the mechanisms of viral transmission are difficult to distinguish. Here we present a cross-platform software framework called "Infectio," which is capable of simulating transmission phenotypes in tissue culture of virtually any virus. Infectio can estimate interdependent biological parameters, for example for vaccinia virus infection, and differentiate between cell-cell and cell-free virus spreading. Infectio assists in elucidating virus transmission mechanisms, a feature useful for designing strategies of perturbing or enhancing viral transmission. The complexity of the Infectio software is low compared to that of other software commonly used to quantitate features of cell biological images, which yields stable and relatively error-free output from Infectio. The software is open source (GPLv3 license), and operates on the major platforms (Windows, Mac, and Linux). The complete source code can be downloaded from http://infectio.github.io/index.html. IMPORTANCE Infectio presents a generalized platform to analyze virus infection spread between cells. It allows the simulation of plaque phenotypes from image-based assays. Viral plaques are the result of virus spreading from primary infected cells to neighboring cells. This is a complex process and involves neighborhood effects at cell-cell contact sites or fluid dynamics in the extracellular medium. Infectio differentiates between two major modes of virus transmission between cells, allowing in silico testing of hypotheses about spreading

  7. Queen pheromones: The chemical crown governing insect social life.

    PubMed

    Holman, Luke

    2010-11-01

    Group-living species produce signals that alter the behavior and even the physiology of their social partners. Social insects possess especially sophisticated chemical communication systems that govern every aspect of colony life, including the defining feature of eusociality: reproductive division of labor. Current evidence hints at the central importance of queen pheromones, but progress has been hindered by the fact that such pheromones have only been isolated in honeybees. In a pair of papers on the ant Lasius niger, we identified and investigated a queen pheromone regulating worker sterility. The cuticular hydrocarbon 3-methylhentriacontane (3-MeC(31)) is correlated with queen maturity and fecundity and workers are also more likely to execute surplus queens that have low amounts of this chemical. Experiments with synthetic 3-MeC(31) found that it inhibits ovarian development in queenless workers and lowers worker aggression towards objects coated with it. Production of 3-MeC(31) by queens was depressed by an experimental immune challenge, and the same chemical was abundant on queenlaid eggs, suggesting that the workers' responses to the queen are conditional on her health and fecundity. Together with other studies, these results indicate that queen pheromones are honest signals of quality that simultaneously regulate multiple social behaviors. PMID:21331238

  8. Bat airway epithelial cells: a novel tool for the study of zoonotic viruses.

    PubMed

    Eckerle, Isabella; Ehlen, Lukas; Kallies, René; Wollny, Robert; Corman, Victor M; Cottontail, Veronika M; Tschapka, Marco; Oppong, Samuel; Drosten, Christian; Müller, Marcel A

    2014-01-01

    Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells. PMID:24454736

  9. Bat Airway Epithelial Cells: A Novel Tool for the Study of Zoonotic Viruses

    PubMed Central

    Eckerle, Isabella; Ehlen, Lukas; Kallies, René; Wollny, Robert; Corman, Victor M.; Cottontail, Veronika M.; Tschapka, Marco; Oppong, Samuel; Drosten, Christian; Müller, Marcel A.

    2014-01-01

    Bats have been increasingly recognized as reservoir of important zoonotic viruses. However, until now many attempts to isolate bat-borne viruses in cell culture have been unsuccessful. Further, experimental studies on reservoir host species have been limited by the difficulty of rearing these species. The epithelium of the respiratory tract plays a central role during airborne transmission, as it is the first tissue encountered by viral particles. Although several cell lines from bats were established recently, no well-characterized, selectively cultured airway epithelial cells were available so far. Here, primary cells and immortalized cell lines from bats of the two important suborders Yangochiroptera and Yinpterochiroptera, Carollia perspicillata (Seba's short-tailed bat) and Eidolon helvum (Straw-colored fruit bat), were successfully cultured under standardized conditions from both fresh and frozen organ specimens by cell outgrowth of organ explants and by the use of serum-free primary cell culture medium. Cells were immortalized to generate permanent cell lines. Cells were characterized for their epithelial properties such as expression of cytokeratin and tight junctions proteins and permissiveness for viral infection with Rift-Valley fever virus and vesicular stomatitis virus Indiana. These cells can serve as suitable models for the study of bat-borne viruses and complement cell culture models for virus infection in human airway epithelial cells. PMID:24454736

  10. Connections matter − how viruses use cell–cell adhesion components

    PubMed Central

    Mateo, Mathieu; Generous, Alex; Sinn, Patrick L.; Cattaneo, Roberto

    2015-01-01

    ABSTRACT The epithelium is a highly organized type of animal tissue. Except for blood and lymph vessels, epithelial cells cover the body, line its cavities in single or stratified layers and support exchange between compartments. In addition, epithelia offer to the body a barrier to pathogen invasion. To transit through or to replicate in epithelia, viruses have to face several obstacles, starting from cilia and glycocalyx where they can be neutralized by secreted immunoglobulins. Tight junctions and adherens junctions also prevent viruses to cross the epithelial barrier. However, viruses have developed multiple strategies to blaze their path through the epithelium by utilizing components of cell–cell adhesion structures as receptors. In this Commentary, we discuss how viruses take advantage of the apical junction complex to spread. Whereas some viruses quickly disrupt epithelium integrity, others carefully preserve it and use cell adhesion proteins and their cytoskeletal connections to rapidly spread laterally. This is exemplified by the hidden transmission of enveloped viruses that use nectins as receptors. Finally, several viruses that replicate preferentially in cancer cells are currently used as experimental cancer therapeutics. Remarkably, these viruses use cell adhesion molecules as receptors, probably because – to reach tumors and metastases – oncolytic viruses must efficiently traverse or break epithelia. PMID:26046138

  11. Structural Co-Evolution of Viruses and Cells in the Primordial World

    NASA Astrophysics Data System (ADS)

    Jalasvuori, Matti; Bamford, Jaana K. H.

    2008-04-01

    Viruses and cells co-evolve due to the parasitic nature of viruses. Yet there are no models suggesting how the unicellular organisms and their viruses might co-evolve structurally. Here, in this study, we plunge into this unexplored field from a wide perspective and try to describe some of the intriguing ways in which viruses may have shaped the cellular life forms on the ancient Earth. At first we propose a scenario where viruses act as a driving force in the emergence of bacterial cell walls by providing favorable intermediates for the otherwise improbable steps in the cell wall generation. We also discuss the role of viruses in the evolution of cell surface components such as receptors and second membranes. Finally we focus on hypothetical proto-viruses, the selfish abusers of the RNA-world, in explaining some of the very early stages in the origin and evolution of life. Proto-viruses may be responsible for creating the first true cells in order to support their selfish needs. In this model we also suggest a logical pathway to explaining the emergence of modern viruses.

  12. Id3 Controls Cell Death of 2B4+ Virus-Specific CD8+ T Cells in Chronic Viral Infection.

    PubMed

    Menner, Alexandra J; Rauch, Katharina S; Aichele, Peter; Pircher, Hanspeter; Schachtrup, Christian; Schachtrup, Kristina

    2015-09-01

    Sustained Ag persistence in chronic infection results in a deregulated CD8(+) T cell response that is characterized by T cell exhaustion and cell death of Ag-specific CD8(+) T cells. Yet, the underlying transcriptional mechanisms regulating CD8(+) T cell exhaustion and cell death are poorly defined. Using the experimental mouse model of lymphocytic choriomeningitis virus infection, we demonstrate that the transcriptional regulator Id3 controls cell death of virus-specific CD8(+) T cells in chronic infection. By comparing acute and chronic infection, we showed that Id3 (-) virus-specific CD8(+) T cells were less abundant, whereas the absolute numbers of Id3 (+) virus-specific CD8(+) T cells were equal in chronic and acute infection. Phenotypically, Id3 (-) and Id3 (+) cells most prominently differed with regard to expression of the surface receptor 2B4; although Id3 (-) cells were 2B4(+), almost all Id3 (+) cells lacked expression of 2B4. Lineage-tracing experiments showed that cells initially expressing Id3 differentiated into Id3 (-)2B4(+) cells; in turn, these cells were terminally differentiated and highly susceptible to cell death under conditions of persisting Ag. Enforced Id3 expression specifically increased the persistence of 2B4(+) virus-specific CD8(+) T cells by decreasing susceptibility to Fas/Fas ligand-mediated cell death. Thus, our findings reveal that the transcriptional regulator Id3 promotes the survival of virus-specific CD8(+) T cells in chronic infection and suggest that targeting Id3 might be beneficial for preventing cell death of CD8(+) T cells in chronic infection or for promoting cell death of uncontrolled, hyperactive CD8(+) T cells to prevent immunopathology. PMID:26232435

  13. Examining Human T-Lymphotropic Virus Type 1 Infection and Replication by Cell-Free Infection with Recombinant Virus Vectors

    PubMed Central

    Derse, David; Hill, Shawn A.; Lloyd, Patricia A.; Chung, Hye-kyung; Morse, Barry A.

    2001-01-01

    A sensitive and quantitative cell-free infection assay, utilizing recombinant human T-cell leukemia virus type 1 (HTLV-1)-based vectors, was developed in order to analyze early events in the virus replication cycle. Previous difficulties with the low infectivity and restricted expression of the virus have prevented a clear understanding of these events. Virus stocks were generated by transfecting cells with three plasmids: (i) a packaging plasmid encoding HTLV-1 structural and regulatory proteins, (ii) an HTLV-1 transfer vector containing either firefly luciferase or enhanced yellow fluorescent protein genes, and (iii) an envelope expression plasmid. Single-round infections were initiated by exposing target cells to filtered supernatants and quantified by assaying for luciferase activity in cell extracts or by enumerating transduced cells by flow cytometry. Transduction was dependent on reverse transcription and integration of the recombinant virus genome, as shown by the effects of the reverse transcriptase inhibitor 3′-azido-3′-deoxythymidine (AZT) and by mutation of the integrase gene in the packaging vector, respectively. The 50% inhibitory concentration of AZT was determined to be 30 nM in this HTLV-1 replication system. The stability of HTLV-1 particles, pseudotyped with either vesicular stomatitis virus G protein or HTLV-1 envelope, was typical of retroviruses, exhibiting a half-life of approximately 3.5 h at 37°C. The specific infectivity of recombinant HTLV-1 virions was at least 3 orders of magnitude lower than that of analogous HIV-1 particles, though both were pseudotyped with the same envelope. Thus, the low infectivity of HTLV-1 is determined in large part by properties of the core particle and by the efficiency of postentry processes. PMID:11507191

  14. Full-Length GB Virus C (Hepatitis G Virus) RNA Transcripts Are Infectious in Primary CD4-Positive T Cells

    PubMed Central

    Xiang, Jinhua; Wünschmann, Sabina; Schmidt, Warren; Shao, Jianqiang; Stapleton, Jack T.

    2000-01-01

    GB virus C (GBV-C or hepatitis G virus) is a recently described flavivirus which frequently leads to chronic viremia in humans. Although GBV-C is associated with acute posttransfusion hepatitis, it is not clear if the virus is pathogenic for humans. We constructed a full-length cDNA from the plasma of a person with chronic GBV-C viremia. Peripheral blood mononuclear cells (PBMCs) transfected with full-length RNA transcripts from this GBV-C clone resulted in viral replication. This was demonstrated by serial passage of virus from cell culture supernatants, detection of increasing concentrations of positive- and negative-sense GBV-C RNA over time, and the detection of the GBV-C E2 antigen by confocal microscopy. In addition, two types of GBV-C particles were identified in cell lysates; these particles had buoyant densities of 1.06 and 1.12 to 1.17 g/ml in sucrose gradients. PBMCs sorted for expression of CD4 contained 100-fold-more GBV-C RNA than CD4-negative cells. Taken together, these data demonstrate that RNA transcripts from GBV-C full-length cDNA are infectious in primary CD4-positive T cells. In contrast, RNA transcripts from an infectious hepatitis C virus clone did not replicate in the same cell culture system. Infectious RNA transcripts from GBV-C cDNA should prove useful for studying viral replication and may allow identification of differences between GBV-C and hepatitis C virus cultivation in vitro. PMID:10982359

  15. Porcine macrophage Cdelta2+ and Cdelta2- cell lines support influenza virus infection and replication and Cdelta2+ cells mount innate immune responses to influenza virus infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Respiratory epithelial cells are the first cells which are infected with influenza virus and these cells play a major role in influenza pathogenesis. However, many studies have shown that alveolar macrophages also play a very important role in the pathogenesis and immunity to influenza infection. Un...

  16. Virus and Bacterial Cell Chemical Analysis by NanoSIMS

    SciTech Connect

    Weber, P; Holt, J

    2008-07-28

    In past work for the Department of Homeland Security, the LLNL NanoSIMS team has succeeded in extracting quantitative elemental composition at sub-micron resolution from bacterial spores using nanometer-scale secondary ion mass spectrometry (NanoSIMS). The purpose of this task is to test our NanoSIMS capabilities on viruses and bacterial cells. This initial work has proven successful. We imaged Tobacco Mosaic Virus (TMV) and Bacillus anthracis Sterne cells using scanning electron microscopy (SEM) and then analyzed those samples by NanoSIMS. We were able resolve individual viral particles ({approx}18 nm by 300 nm) in the SEM and extract correlated elemental composition in the NanoSIMS. The phosphorous/carbon ratio observed in TMV is comparable to that seen in bacterial spores (0.033), as was the chlorine/carbon ratio (0.11). TMV elemental composition is consistent from spot to spot, and TMV is readily distinguished from debris by NanoSIMS analysis. Bacterial cells were readily identified in the SEM and relocated in the NanoSIMS for elemental analysis. The Ba Sterne cells were observed to have a measurably lower phosphorous/carbon ratio (0.005), as compared to the spores produced in the same run (0.02). The chlorine/carbon ratio was approximately 2.5X larger in the cells (0.2) versus the spores (0.08), while the fluorine/carbon ratio was approximately 10X lower in the cells (0.008) than the spores (0.08). Silicon/carbon ratios for both cells and spores encompassed a comparable range. The initial data in this study suggest that high resolution analysis is useful because it allows the target agent to be analyzed separate from particulates and other debris. High resolution analysis would also be useful for trace sample analysis. The next step in this work is to determine the potential utility of elemental signatures in these kinds of samples. We recommend bulk analyses of media and agent samples to determine the range of media compositions in use, and to determine how

  17. Several Human Liver Cell Expressed Apolipoproteins Complement HCV Virus Production with Varying Efficacy Conferring Differential Specific Infectivity to Released Viruses

    PubMed Central

    Doepke, Mandy; Vieyres, Gabrielle; Todt, Daniel; Wölk, Benno; Vondran, Florian W. R.; Geffers, Robert; Lauber, Chris; Kaderali, Lars; Penin, François; Pietschmann, Thomas

    2015-01-01

    Apolipoprotein E (ApoE), an exchangeable apolipoprotein, is necessary for production of infectious Hepatitis C virus (HCV) particles. However, ApoE is not the only liver-expressed apolipoprotein and the role of other apolipoproteins for production of infectious HCV progeny is incompletely defined. Therefore, we quantified mRNA expression of human apolipoproteins in primary human hepatocytes. Subsequently, cDNAs encoding apolipoproteins were expressed in 293T/miR-122 cells to explore if they complement HCV virus production in cells that are non-permissive due to limiting endogenous levels of human apolipoproteins. Primary human hepatocytes expressed high mRNA levels of ApoA1, A2, C1, C3, E, and H. ApoA4, A5, B, D, F, J, L1, L2, L3, L4, L6, M, and O were expressed at intermediate levels, and C2, C4, and L5 were not detected. All members of the ApoA and ApoC family of lipoproteins complemented HCV virus production in HCV transfected 293T/miR-122 cells, albeit with significantly lower efficacy compared with ApoE. In contrast, ApoD expression did not support production of infectious HCV. Specific infectivity of released particles complemented with ApoA family members was significantly lower compared with ApoE. Moreover, the ratio of extracellular to intracellular infectious virus was significantly higher for ApoE compared to ApoA2 and ApoC3. Since apolipoproteins complementing HCV virus production share amphipathic alpha helices as common structural features we altered the two alpha helices of ApoC1. Helix breaking mutations in both ApoC1 helices impaired virus assembly highlighting a critical role of alpha helices in apolipoproteins supporting HCV assembly. In summary, various liver expressed apolipoproteins with amphipathic alpha helices complement HCV virus production in human non liver cells. Differences in the efficiency of virus assembly, the specific infectivity of released particles, and the ratio between extracellular and intracellular infectivity point to

  18. Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

    SciTech Connect

    Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro

    2011-05-25

    Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-{gamma} signaling pathway via inhibition of phosphorylated STAT1 dimerization.

  19. Mandibular gland components of european and africanized honey bee queens (Apis mellifera L.).

    PubMed

    Pankiw, T; Winston, M L; Plettner, E; Slessor, K N; Pettis, J S; Taylor, O R

    1996-04-01

    The composition of the five-component honey bee queen mandibular gland pheromone (QMP) of mated European honey bee queens was compared to those of virgin and drone-laying (i.e., laying only haploid unfertilized eggs that develop into males), European queens and Africanized mated queens. QMP of mated European queens showed significantly greater quantities of individual components than all queen types compared, except for a significantly greater quantity of 9-hydroxy-(E)-2-decenoic acid (9-HDA) found in Africanized queens. Glands of European drone-laying queens contained quantities intermediate between virgin and mated queens, reflecting their intermediate reproductive state and age. QMP ontogeny shifts from a high proportion of 9-keto-(E)-2-decenoic acid (ODA) in young unmated queens to roughly equal proportions of ODA and 9-HDA in mated queens. A biosynthetic shift occurs after mating that results in a greater proportion of 9-HDA, methylp-hydroxybenzoate (HOB), and 4-hydroxy-3-methoxyphenylethanol (HVA) production, accompanied by a decreased proportion of ODA. Africanized QMP proportions of ODA and 9-HDA were significantly different from European queens. A quantitative definition of a "queen equivalent" of QMP is proposed for the various queen types, and a standard queen equivalent for mated European honeybee queen mandibular gland pheromone is adopted as 200µg ODA, 80µg 9-HDA, 20µg HOB, and 2 µg HVA. PMID:24227572

  20. Susceptibility of Human Embryonic Stem Cell-Derived Neural Cells to Japanese Encephalitis Virus Infection

    PubMed Central

    Shen, Shih-Cheng; Shen, Ching-I; Lin, Ho; Chen, Chun-Jung; Chang, Chia-Yu; Chen, Sheng-Mei; Lee, Hsiu-Chin; Lai, Ping-Shan; Su, Hong-Lin

    2014-01-01

    Pluripotent human embryonic stem cells (hESCs) can be efficiently directed to become immature neuroepithelial precursor cells (NPCs) and functional mature neural cells, including neurotransmitter-secreting neurons and glial cells. Investigating the susceptibility of these hESCs-derived neural cells to neurotrophic viruses, such as Japanese encephalitis virus (JEV), provides insight into the viral cell tropism in the infected human brain. We demonstrate that hESC-derived NPCs are highly vulnerable to JEV infection at a low multiplicity of infection (MOI). In addition, glial fibrillary acid protein (GFAP)-expressing glial cells are also susceptible to JEV infection. In contrast, only a few mature neurons were infected at MOI 10 or higher on the third day post-infection. In addition, functional neurotransmitter-secreting neurons are also resistant to JEV infection at high MOI. Moreover, we discover that vimentin intermediate filament, reported as a putative neurovirulent JEV receptor, is highly expressed in NPCs and glial cells, but not mature neurons. These results indicate that the expression of vimentin in neural cells correlates to the cell tropism of JEV. Finally, we further demonstrate that membranous vimentin is necessary for the susceptibility of hESC-derived NPCs to JEV infection. PMID:25517725

  1. Measles virus was present in the inner cell of the acrosyringium in the skin rash.

    PubMed

    Yanagihara, M; Fujii, T; Mochizuki, T; Ishizaki, H; Sata, T

    1998-01-01

    A case of measles in a 26-year-old Japanese man is reported. A skin specimen taken on the third eruptive day from a maculopapular eruption on his chest was immunohistopathologically and electron microscopically examined using a rabbit polyclonal antibody against the nucleocapsid protein of the measles virus. The measles virus antigen was found in the inner cells of the acrosyringium and hair follicles. The measles virus nucleocapsid was electron microscopically identified in the nuclei of the inner cells of the acrosyringium. The findings suggest that the sweat from skin lesions might contain the measles virus. PMID:9875969

  2. p53-Mediated Cellular Response to DNA Damage in Cells with Replicative Hepatitis B Virus

    NASA Astrophysics Data System (ADS)

    Puisieux, Alain; Ji, Jingwei; Guillot, Celine; Legros, Yann; Soussi, Thierry; Isselbacher, Kurt; Ozturk, Mehmet

    1995-02-01

    Wild-type p53 acts as a tumor suppressor gene by protecting cells from deleterious effects of genotoxic agents through the induction of a G_1/S arrest or apoptosis as a response to DNA damage. Transforming proteins of several oncogenic DNA viruses inactivate tumor suppressor activity of p53 by blocking this cellular response. To test whether hepatitis B virus displays a similar effect, we studied the p53-mediated cellular response to DNA damage in 2215 hepatoma cells with replicative hepatitis B virus. We demonstrate that hepatitis B virus replication does not interfere with known cellular functions of p53 protein.

  3. Vaccinia virus, herpes simplex virus, and carcinogens induce DNA amplification in a human cell line and support replication of a helpervirus dependent parvovirus

    SciTech Connect

    Schlehofer, J.R.; Ehrbar, M.; zur Hausen, H.

    1986-07-15

    The SV40-transformed human kidney cell line, NB-E, amplifies integrated as well as episomal SV40 DNA upon treatment with chemical (DMBA) or physical (uv irradiation) carcinogens (initiators) as well as after infection with herpes simplex virus (HSV) type 1 or with vaccinia virus. In addition it is shown that vaccinia virus induces SV40 DNA amplification also in the SV40-transformed Chinese hamster embryo cell line, CO631. These findings demonstrate that human cells similar to Chinese hamster cells amplify integrated DNA sequences after treatment with carcinogens or infection with specific viruses. Furthermore, a poxvirus--vaccinia virus--similar to herpes group viruses induces DNA amplification. As reported for other systems, the vaccinia virus-induced DNA amplification in NB-E cells is inhibited by coinfection with adeno-associated virus (AAV) type 5. This is in line with previous studies on inhibition of carcinogen- or HSV-induced DNA amplification in CO631 cells. The experiments also demonstrate that vaccinia virus, in addition to herpes and adenoviruses acts as a helper virus for replication and structural antigen synthesis of AAV-5 in NB-E cells.

  4. Avian Influenza Viruses, Inflammation, and CD8(+) T Cell Immunity.

    PubMed

    Wang, Zhongfang; Loh, Liyen; Kedzierski, Lukasz; Kedzierska, Katherine

    2016-01-01

    Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds, infect domestic poultry, and are capable of causing sporadic bird-to-human transmissions. AIVs capable of infecting humans include a highly pathogenic AIV H5N1, first detected in humans in 1997, and a low pathogenic AIV H7N9, reported in humans in 2013. Both H5N1 and H7N9 cause severe influenza disease in humans, manifested by acute respiratory distress syndrome, multi-organ failure, and high mortality rates of 60% and 35%, respectively. Ongoing circulation of H5N1 and H7N9 viruses in wild birds and poultry, and their ability to infect humans emphasizes their epidemic and pandemic potential and poses a public health threat. It is, thus, imperative to understand the host immune responses to the AIVs so we can control severe influenza disease caused by H5N1 or H7N9 and rationally design new immunotherapies and vaccines. This review summarizes our current knowledge on AIV epidemiology, disease symptoms, inflammatory processes underlying the AIV infection in humans, and recent studies on universal pre-existing CD8(+) T cell immunity to AIVs. Immune responses driving the host recovery from AIV infection in patients hospitalized with severe influenza disease are also discussed. PMID:26973644

  5. Avian Influenza Viruses, Inflammation, and CD8+ T Cell Immunity

    PubMed Central

    Wang, Zhongfang; Loh, Liyen; Kedzierski, Lukasz; Kedzierska, Katherine

    2016-01-01

    Avian influenza viruses (AIVs) circulate naturally in wild aquatic birds, infect domestic poultry, and are capable of causing sporadic bird-to-human transmissions. AIVs capable of infecting humans include a highly pathogenic AIV H5N1, first detected in humans in 1997, and a low pathogenic AIV H7N9, reported in humans in 2013. Both H5N1 and H7N9 cause severe influenza disease in humans, manifested by acute respiratory distress syndrome, multi-organ failure, and high mortality rates of 60% and 35%, respectively. Ongoing circulation of H5N1 and H7N9 viruses in wild birds and poultry, and their ability to infect humans emphasizes their epidemic and pandemic potential and poses a public health threat. It is, thus, imperative to understand the host immune responses to the AIVs so we can control severe influenza disease caused by H5N1 or H7N9 and rationally design new immunotherapies and vaccines. This review summarizes our current knowledge on AIV epidemiology, disease symptoms, inflammatory processes underlying the AIV infection in humans, and recent studies on universal pre-existing CD8+ T cell immunity to AIVs. Immune responses driving the host recovery from AIV infection in patients hospitalized with severe influenza disease are also discussed. PMID:26973644

  6. Pathogenic Chikungunya Virus Evades B Cell Responses to Establish Persistence.

    PubMed

    Hawman, David W; Fox, Julie M; Ashbrook, Alison W; May, Nicholas A; Schroeder, Kristin M S; Torres, Raul M; Crowe, James E; Dermody, Terence S; Diamond, Michael S; Morrison, Thomas E

    2016-08-01

    Chikungunya virus (CHIKV) and related alphaviruses cause epidemics of acute and chronic musculoskeletal disease. To investigate the mechanisms underlying the failure of immune clearance of CHIKV, we studied mice infected with an attenuated CHIKV strain (181/25) and the pathogenic parental strain (AF15561), which differ by five amino acids. Whereas AF15561 infection of wild-type mice results in viral persistence in joint tissues, 181/25 is cleared. In contrast, 181/25 infection of μMT mice lacking mature B cells results in viral persistence in joint tissues, suggesting that virus-specific antibody is required for clearance of infection. Mapping studies demonstrated that a highly conserved glycine at position 82 in the A domain of the E2 glycoprotein impedes clearance and neutralization of multiple CHIKV strains. Remarkably, murine and human antibodies targeting E2 domain B failed to neutralize pathogenic CHIKV strains efficiently. Our data suggest that pathogenic CHIKV strains evade E2 domain-B-neutralizing antibodies to establish persistence. PMID:27452455

  7. Selective Destruction Of Cells Infected With The Human Immunodeficiency Virus

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2006-03-28

    Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a varient of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

  8. Selective destruction of cells infected with human immunodeficiency virus

    DOEpatents

    Keener, William K.; Ward, Thomas E.

    2003-09-30

    Compositions and methods for selectively killing a cell containing a viral protease are disclosed. The composition is a variant of a protein synthesis inactivating toxin wherein a viral protease cleavage site is interposed between the A and B chains. The variant of the type II ribosome-inactivating protein is activated by digestion of the viral protease cleavage site by the specific viral protease. The activated ribosome-inactivating protein then kills the cell by inactivating cellular ribosomes. A preferred embodiment of the invention is specific for human immunodeficiency virus (HIV) and uses ricin as the ribosome-inactivating protein. In another preferred embodiment of the invention, the variant of the ribosome-inactivating protein is modified by attachment of one or more hydrophobic agents. The hydrophobic agent facilitates entry of the variant of the ribosome-inactivating protein into cells and can lead to incorporation of the ribosome-inactivating protein into viral particles. Still another preferred embodiment of the invention includes a targeting moiety attached to the variants of the ribosome-inactivating protein to target the agent to HIV infectable cells.

  9. Virus and Cell RNAs Expressed during Epstein-Barr Virus Replication

    PubMed Central

    Yuan, Jing; Cahir-McFarland, Ellen; Zhao, Bo; Kieff, Elliott

    2006-01-01

    Changes in Epstein-Barr virus (EBV) and cell RNA levels were assayed following immunoglobulin G (IgG) cross-linking-induced replication in latency 1-infected Akata Burkitt B lymphoblasts. EBV replication as assayed by membrane gp350 expression was ∼5% before IgG cross-linking and increased to more than 50% 48 h after induction. Seventy-two hours after IgG cross-linking, gp350-positive cells excluded propidium iodide as well as gp350-negative cells. EBV RNA levels changed temporally in parallel with previously defined sensitivity to inhibitors of protein or viral DNA synthesis. BZLF1 immediate-early RNA levels doubled by 2 h and reached a peak at 4 h, whereas BMLF1 doubled by 4 h with a peak at 8 h, and BRLF1 doubled by 8 h with peak at 12 h. Early RNAs peaked at 8 to 12 h, and late RNAs peaked at 24 h. Hybridization to intergenic sequences resulted in evidence for new EBV RNAs. Surprisingly, latency III (LTIII) RNAs for LMP1, LMP2, EBNALP, EBNA2, EBNA3A, EBNA3C, and BARTs were detected at 8 to 12 h and reached maxima at 24 to 48 h. EBNA2 and LMP1 were at full LTIII levels by 48 h and localized to gp350-positive cells. Thus, LTIII expression is a characteristic of late EBV replication in both B lymphoblasts and epithelial cells in immune-comprised people (J. Webster-Cyriaque, J. Middeldorp, and N. Raab-Traub, J. Virol. 74:7610-7618, 2000). EBV replication significantly altered levels of 401 Akata cell RNAs, of which 122 RNAs changed twofold or more relative to uninfected Akata cells. Mitogen-activated protein kinase levels were significantly affected. Late expression of LTIII was associated with induction of NF-κB responsive genes including IκBα and A20. The exclusion of propidium, expression of EBV LTIII RNAs and proteins, and up-regulation of specific cell RNAs are indicative of vital cell function late in EBV replication. PMID:16474161

  10. Guiding plant virus particles to integrin-displaying cells

    NASA Astrophysics Data System (ADS)

    Hovlid, Marisa L.; Steinmetz, Nicole F.; Laufer, Burkhardt; Lau, Jolene L.; Kuzelka, Jane; Wang, Qian; Hyypiä, Timo; Nemerow, Glen R.; Kessler, Horst; Manchester, Marianne; Finn, M. G.

    2012-05-01

    Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity for several different cancer cell lines that express RGD-binding integrin receptors.Viral nanoparticles (VNPs) are structurally regular, highly stable, tunable nanomaterials that can be conveniently produced in high yields. Unmodified VNPs from plants and bacteria generally do not show tissue specificity or high selectivity in binding to or entry into mammalian cells. They are, however, malleable by both genetic and chemical means, making them useful scaffolds for the display of large numbers of cell- and tissue-targeting ligands, imaging moieties, and/or therapeutic agents in a well-defined manner. Capitalizing on this attribute, we modified the genetic sequence of the Cowpea mosaic virus (CPMV) coat protein to display an RGD oligopeptide sequence derived from human adenovirus type 2 (HAdV-2). Concurrently, wild-type CPMV was modified via NHS acylation and Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) chemistry to attach an integrin-binding cyclic RGD peptide. Both types of particles showed strong and selective affinity

  11. Role of the cytoskeleton in cell-to-cell transmission of human immunodeficiency virus.

    PubMed Central

    Pearce-Pratt, R; Malamud, D; Phillips, D M

    1994-01-01

    We previously observed that when human immunodeficiency virus (HIV)-infected T lymphocytes are added to epithelial cells, they adhere, polarize, and secrete virions unidirectionally onto the epithelium. Epithelial cells subsequently take up virus and become productively infected. We report here that colchicine treatment of T-lymphocyte suspensions induced lymphocyte polarization, redistribution of F-actin into a pseudopod, and secretion of HIV from the pseudopod. Immobilization of T lymphocytes on negatively charged plastic also caused redistribution of F-actin and unidirectional secretion of HIV onto the plastic. As neither colchicine nor adhesion caused an increase in HIV secretion, they apparently act by focusing secretion to the tip of the pseudopod. We speculate that adhesion-induced polar secretion of HIV, from activated mononuclear cells onto epithelia, is a cytoskeleton-mediated process which may be involved in HIV transmission in vivo. Images PMID:8151760

  12. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    PubMed Central

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-01-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. PMID:15837934

  13. A coiled-coil interaction mediates cauliflower mosaic virus cell-to-cell movement

    NASA Astrophysics Data System (ADS)

    Stavolone, Livia; Villani, Maria Elena; Leclerc, Denis; Hohn, Thomas

    2005-04-01

    The function of the virion-associated protein (VAP) of cauliflower mosaic virus (CaMV) has long been only poorly understood. VAP is associated with the virion but is dispensable for virus morphogenesis and replication. It mediates virus transmission by aphids through simultaneous interaction with both the aphid transmission factor and the virion. However, although insect transmission is not fundamental to CaMV survival, VAP is indispensable for spreading the virus infection within the host plant. We used a GST pull-down technique to demonstrate that VAP interacts with the viral movement protein through coiled-coil domains and surface plasmon resonance to measure the interaction kinetics. We mapped the movement protein coiled-coil to the C terminus of the protein and proved that it self-assembles as a trimer. Immunogold labeling/electron microscopy revealed that the VAP and viral movement protein colocalize on CaMV particles within plasmodesmata. These results highlight the multifunctional potential of the VAP protein conferred by its efficient coiled-coil interaction system and show a plant virus possessing a surface-exposed protein (VAP) mediating viral entry into host cells. movement protein | virion-associated protein | Biacore

  14. Vaccinia Virus Tropism for Primary Hematolymphoid Cells Is Determined by Restricted Expression of a Unique Virus Receptor

    PubMed Central

    Chahroudi, Ann; Chavan, Rahul; Koyzr, Natalia; Waller, Edmund K.; Silvestri, Guido; Feinberg, Mark B.

    2005-01-01

    The presumed broad tropism of poxviruses has stymied attempts to identify both the cellular receptor(s) and the viral determinant(s) for binding. Detailed studies of poxvirus binding to and infection of primary human cells have not been conducted. In particular, the determinants of target cell infection and the consequences of infection for cells involved in the generation of antiviral immune responses are incompletely understood. In this report, we show that vaccinia virus (VV) exhibits a more restricted tropism for primary hematolymphoid human cells than has been previously recognized. We demonstrate that vaccinia virus preferentially infects antigen-presenting cells (dendritic cells, monocytes/macrophages, and B cells) and activated T cells, but not resting T cells. The infection of activated T cells is permissive, with active viral replication and production of infectious progeny. Susceptibility to infection is determined by restricted expression of a cellular receptor that is induced de novo upon T-cell activation and can be removed from the cell surface by either trypsin or pronase treatment. The VV receptor expressed on activated T cells displays unique characteristics that distinguish it from the receptor used to infect cell lines in culture. The observed restricted tropism of VV may have significant consequences for the understanding of natural poxvirus infection and immunity and for poxvirus-based vaccine development. PMID:16051832

  15. Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses.

    PubMed

    Ren, Linzhu; Peng, Zhiyuan; Chen, Xinrong; Ouyang, Hongsheng

    2016-04-01

    Cell-based reporter systems have facilitated studies of viral replication and pathogenesis, virus detection, and drug susceptibility testing. There are three types of cell-based reporter systems that express certain reporter protein for positive-sense single strand RNA virus infections. The first type is classical reporter system, which relies on recombinant virus, reporter virus particle, or subgenomic replicon. During infection with the recombinant virus or reporter virus particle, the reporter protein is expressed and can be detected in real time in a dose-dependent manner. Using subgenomic replicon, which are genetically engineered viral RNA molecules that are capable of replication but incapable of producing virions, the translation and replication of the replicon could be tracked by the accumulation of reporter protein. The second type of reporter system involves genetically engineered cells bearing virus-specific protease cleavage sequences, which can sense the incoming viral protease. The third type is based on viral replicase, which can report the specific virus infection via detection of the incoming viral replicase. This review specifically focuses on the major technical breakthroughs in the design of cell-based reporter systems and the application of these systems to the further understanding and control of viruses over the past few decades. PMID:26728654

  16. Genistein inhibits the replication of avian leucosis virus subgroup J in DF-1 cells.

    PubMed

    Qian, Kun; Gao, Ai-jun; Zhu, Ming-yue; Shao, Hong-xia; Jin, Wen-jie; Ye, Jian-qiang; Qin, Ai-jian

    2014-11-01

    To investigate the antiviral effects of genistein on the replication of avian leukosis virus subgroup J (ALV-J) in DF-1 cells, the cells were treated with genistein at different time points and the antiviral effects were examined by using a variety of assays. We determined that genistein strongly inhibited viral gene expression and decreased the viral protein level in the cell supernatant and the cytoplasm without alerting virus receptor expression and viral attachment. We also observed that genistein was not found to interfere with virus entry, but significantly inhibited both viral gene transcriptions at 24h post infection and virus release, which indicate that genistein exerts its inhibitory effects on the late phase of ALV-J replicative cycle. These results demonstrate that genistein effectively block ALV-J replication by inhibiting virus transcription and release in DF-1 cells, which may be useful for therapeutic drug design. PMID:25197039

  17. [Study on the B cell linear epitopes of rabies virus CVS-11 nucleoprotein].

    PubMed

    Lv, Xin-Jun; Shen, Xin-Xin; Yu, Peng-Cheng; Li, Hao; Wang, Li-Hua; Tang, Qing; Liang, Guo-Dong

    2014-05-01

    To study the B cell linear epitopes of rabies virus CVS-11 nucleoprotein, peptides were synthesized according to the amino acid sequences of B cell linear epitopes. Linear epitopes predicted by bioinformatics analysis were evaluated with immunological techniques. Indirect enzyme-linked immunosorbent assay showed that titers of antibodies to peptides (355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein) were above 1:12 800 in mouse sera. The antibodies recognized denatured rabies virus CVS-11 nucleoprotein in Western blot analysis. Purified anti-peptide antibodies recognized natural rabies virus CVS-11 nucleoprotein in BHK-21 cells in indirect fluorescent antibody test. The 355-369 and 385-400 residues of rabies virus CVS-11 nucleoprotein were validated as B cell linear epitopes. PMID:25118379

  18. BK virus-associated hemorrhagic cystitis after pediatric stem cell transplantation.

    PubMed

    Han, Seung Beom; Cho, Bin; Kang, Jin Han

    2014-12-01

    Hemorrhagic cystitis is a common stem cell transplantation-related complication. The incidence of early-onset hemorrhagic cystitis, which is related to the pretransplant conditioning regimen, has decreased with the concomitant use of mesna and hyperhydration. However, late-onset hemorrhagic cystitis, which is usually caused by the BK virus, continues to develop. Although the BK virus is the most common pathogenic microorganism of poststem cell transplantation late-onset hemorrhagic cystitis, pediatricians outside the hemato-oncology and nephrology specialties tend to be unfamiliar with hemorrhagic cystitis and the BK virus. Moreover, no standard guidelines for the early diagnosis and treatment of BK virus-associated hemorrhagic cystitis after stem cell transplantation have been established. Here, we briefly introduce poststem cell transplantation BK virus-associated hemorrhagic cystitis. PMID:25653684

  19. BK virus-associated hemorrhagic cystitis after pediatric stem cell transplantation

    PubMed Central

    Han, Seung Beom; Kang, Jin Han

    2014-01-01

    Hemorrhagic cystitis is a common stem cell transplantation-related complication. The incidence of early-onset hemorrhagic cystitis, which is related to the pretransplant conditioning regimen, has decreased with the concomitant use of mesna and hyperhydration. However, late-onset hemorrhagic cystitis, which is usually caused by the BK virus, continues to develop. Although the BK virus is the most common pathogenic microorganism of poststem cell transplantation late-onset hemorrhagic cystitis, pediatricians outside the hemato-oncology and nephrology specialties tend to be unfamiliar with hemorrhagic cystitis and the BK virus. Moreover, no standard guidelines for the early diagnosis and treatment of BK virus-associated hemorrhagic cystitis after stem cell transplantation have been established. Here, we briefly introduce poststem cell transplantation BK virus-associated hemorrhagic cystitis. PMID:25653684

  20. Inhibition of Mitosis and Macromolecular Synthesis in Rat Embryo Cells by Kilham Rat Virus

    PubMed Central

    Tennant, Raymond W.

    1971-01-01

    The effects of Kilham rat virus multiplication were studied in cultured rat embryo cells to examine the mechanisms by which virus infection might be related to developmental defects in rats and hamsters. The virus was found to inhibit motosis and deoxyribonucleic acid (DNA) synthesis within 2 to 10 hr after infection. However, total ribonucleic acid synthesis was relatively unaffected until about 20 hr after infection, and total protein synthesis did not decline significantly until loss of viable cells was apparent in the cultures. No effect on chromosomes was detected. The effect of Kilham rat virus on DNA synthesis appears to be due to inhibition of macromolecular synthesis rather than to an inhibition of uptake of precursors into cells. The effect of the virus on mitosis may be an addition to the effect on DNA synthesis, since mitosis is inhibited even in cultures in which cells are able to divide at the time of infection and which have presumably completed DNA synthesis. PMID:5167023

  1. 6K2-induced vesicles can move cell to cell during turnip mosaic virus infection.

    PubMed

    Grangeon, Romain; Jiang, Jun; Wan, Juan; Agbeci, Maxime; Zheng, Huanquan; Laliberté, Jean-François

    2013-01-01

    To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs). However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV) induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked toward the plasma membrane and were associated with plasmodesmata (PD). We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP) to visualize how 6K2 moved intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2. PMID:24409170

  2. Ultrastructure of the Intramandibular Gland of Workers and Queens of the Stingless Bee, Melipona quadrifasciata

    PubMed Central

    Da Cruz-Landim, Carminda; Gracioli-Vitti, Luciana F.; Abdalla, Fábio C.

    2011-01-01

    The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste. PMID:22220493

  3. Effect of larval food amount on ovariole development in queens of Trigona spinipes (Hymenoptera, Apinae).

    PubMed

    Lisboa, L C O; Serrão, J E; Cruz-Landim, C; Campos, L A O

    2005-06-01

    Caste determination in Trigona spinipes Fabricius (Hymenoptera, Apidae, Meliponini) is trophogenic. Larvae that eat about 360 microl of food become queens, while those who consume 36 microl develop into workers. We studied the effect of larval nutrition on the number and length of ovarioles and on ovarian development in fifth instar larvae, white eyed, pink eyed and black-eyed pupae as well as newly emerged adults. All larvae have four ovarioles per ovary, while in queen pupae this number ranged from 8 to 15. Cyst formation, the cell death and other characteristics of ovary morphogenesis were the same regardless of the quantity of food consumed. These results are discussed in relation to caste differentiation in other bees. PMID:15929734

  4. Ultrastructure of the intramandibular gland of workers and queens of the stingless bee, Melipona quadrifasciata.

    PubMed

    Da Cruz-Landim, Carminda; Gracioli-Vitti, Luciana F; Abdalla, Fábio C

    2011-01-01

    The intramandibular glands of workers and queens of Melipona quadrifasciata Lepeletier (Hymenoptera: Apidae), at different ages and from different functional groups, were studied using light and transmission electron microscopy. The results demonstrated that these glands are composed of two types of secretory structures: 1.A hypertrophied epidermis on the dorsal side of the mandible that is an epithelial gland. 2. Free secretory cells filling the inner spaces of the appendices that constitute a unicellular gland. The epithelial gland is larger in the young (1-2-day-old workers), and the gland becomes involuted during the nurse worker stage. The unicellular glands of the workers posses some secretion during all of the studied phases, but secretory activity is more intensive in the foraging workers. Vesicles of secretion are absent in the unicellular glands of queens. These results demonstrate that these glands show functional adaptations in different castes corresponding to the functions of each caste. PMID:22220493

  5. Micro-Raman spectroscopy study of ALVAC virus infected chicken embryo cells

    NASA Astrophysics Data System (ADS)

    Misra, Anupam K.; Kamemoto, Lori E.; Hu, Ningjie; Dykes, Ava C.; Yu, Qigui; Zinin, Pavel V.; Sharma, Shiv K.

    2011-05-01

    Micro- Raman spectroscopic investigation of ALVAC virus and of normal chicken embryo fibroblast cells and the cells infected with ALVAC virus labeled with green fluorescence protein (GFP) were performed with a 785 nm laser. Good quality Micro-Raman spectra of the Alvac II virus were obtained. These spectra show that the ALVAC II virus contains buried tyrosine residues and the coat protein of the virus has α-helical structure. A comparison of Raman spectra of normal and virus infected chicken embryo fibroblast cells revealed that the virus infected cells show additional bands at 535, 928, and 1091 cm-1, respectively, corresponding to δ(C-O-C) glycosidic ring, protein α-helix, and DNA (O-P-O) modes. In addition, the tyrosine resonance double (833 and 855 cm-1) shows reversal in the intensity of the higher-frequency band as compared to the normal cells that can be used to identify the infected cells. In the C-H stretching region, the infected cells show bands with higher intensity as compared to that of the corresponding bands in the normal cells. We also found that the presence of GFP does not affect the Raman spectra of samples when using a 785 nm micro-Raman system because the green fluorescence wavelength of GFP is well below the Stokes-Raman shifted spectral region.

  6. DIESEL EXHAUST ENHANCES INFLUENZA VIRUS INFECTIONS IN RESPIRATORY EPITHELIAL CELLS

    EPA Science Inventory

    Several factors, such as age and nutritional status can affect the susceptibility to influenza infections. Moreover, exposure to air pollutants, such as diesel exhaust (DE), has been shown to affect respiratory virus infections in rodent models. Influenza virus primarily infects ...

  7. The Exonuclease Domain of Lassa Virus Nucleoprotein Is Involved in Antigen-Presenting-Cell-Mediated NK Cell Responses

    PubMed Central

    Russier, Marion; Reynard, Stéphanie; Carnec, Xavier

    2014-01-01

    ABSTRACT Lassa virus is an Old World Arenavirus which causes Lassa hemorrhagic fever in humans, mostly in West Africa. Lassa fever is an important public health problem, and a safe and effective vaccine is urgently needed. The infection causes immunosuppression, probably due to the absence of activation of antigen-presenting cells (dendritic cells and macrophages), low type I interferon (IFN) production, and deficient NK cell function. However, a recombinant Lassa virus carrying D389A and G392A substitutions in the nucleoprotein that abolish the exonuclease activity and IFN activation loses its inhibitory activity and induces strong type I IFN production by dendritic cells and macrophages. We show here that during infection by this mutant Lassa virus, antigen-presenting cells trigger efficient human NK cell responses in vitro, including production of IFN-γ and cytotoxicity. NK cell activation involves close contact with both antigen-presenting cells and soluble factors. We report that infected dendritic cells and macrophages express the NKG2D ligands major histocompatibility complex (MHC) class I-related chains A and B and that they may produce interleukin-12 (IL-12), IL-15, and IL-18, all involved in NK cell functions. NK cell degranulation is significantly increased in cocultures, suggesting that NK cells seem to kill infected dendritic cells and macrophages. This work confirms the inhibitory function of Lassa virus nucleoprotein. Importantly, we demonstrate for the first time that Lassa virus nucleoprotein is involved in the inhibition of antigen-presenting cell-mediated NK cell responses. IMPORTANCE The pathogenesis and immune responses induced by Lassa virus are poorly known. Recently, an exonuclease domain contained in the viral nucleoprotein has been shown to be able to inhibit the type I IFN response by avoiding the recognition of viral RNA by cell sensors. Here, we studied the responses of NK cells to dendritic cells and macrophages infected with a

  8. Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus.

    PubMed Central

    Katz, J M; Wang, M; Webster, R G

    1990-01-01

    When influenza (H3N2) viruses from infected individuals are grown in embryonated chicken eggs, viruses are isolated which differ antigenically and structurally from viruses grown in mammalian Madin-Darby canine kidney (MDCK) cell culture [G.C. Schild, J.S. Oxford, J.C. de Jong, and R.G. Webster, Nature (London) 303:706-709, 1983]. To determine which of these viruses is most representative of virus replicating in the infected individual, a region of the HA gene of virus present in original clinical samples was amplified by using the polymerase chain reaction and sequenced directly. Comparison of 170 amino acid residues of HA1 flanking and containing the receptor-binding site and antigenic sites indicated that over this region, the HA of virus replicating in the infected individual was identical to that of virus after growth in MDCK cells and was distinct from the HA of viruses grown in eggs. Therefore, cultivation of human influenza H3N2 virus in mammalian MDCK cells results in a virus similar to the predominant population of virus found in the infected individual. PMID:2319652

  9. Salmonid fish viruses and cell interactions at early steps of the infective cycle.

    PubMed

    de las Heras, A I; Rodríguez Saint-Jean, S; Pérez-Prieto, S I

    2008-07-01

    A flow cytometric virus-binding assay that directly visualizes the binding and entry of infectious pancreatic necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and virus haemorrhagic septicaemia virus (VHSV) to several cell lines was established. The highest efficiency of binding was shown by the BF-2 cell line and this was used to study, at the attachment level, the interactions of these cells with salmonid fish viruses in coinfections, and to further determine if the earliest stage of the viral growth cycle could explain the previously described loss of infectivity of IHNV when IPNV is present. Our results demonstrated that IPNV binds to around 88% of cells either in single or dual infections, whereas IHNV attachment always decreased in the presence of any of the other viruses. VHSV binding was not affected by IPNV, but coinfection with IHNV reduced the percentage of virus-binding cells, which suggests competition for viral receptors or co-receptors. Internalization of the adsorbed IHNV was not decreased by coinfection with IPNV, so the hypothetical competence could be restricted to the binding step. Treatment of the cells with antiviral agents, such as amantadine or chloroquine, did not affect the binding of IPNV and VHSV, but reduced IHNV binding by more than 30%. Tributylamine affected viral binding of the three viruses to different degrees and inhibited IPNV or IHNV entry in a large percentage of cells treated for 30 min. Tributylamine also inhibited IHNV cytopathic effects in a dose-dependent manner, decreasing the virus yield by 4 log of the 50% endpoint titre, at 10 mm concentration. IPNV was also inhibited, but at a lower level. The results of this study support the hypothesis that IHNV, in contrast to VHSV or IPNV, is less efficient at completing its growth cycle in cells with a simultaneous infection with IPNV. It can be affected at several stages of viral infection and is more sensitive to the action of antiviral compounds. PMID

  10. Apoptosis transcriptional mechanism of feline infectious peritonitis virus infected cells.

    PubMed

    Shuid, Ahmad Naqib; Safi, Nikoo; Haghani, Amin; Mehrbod, Parvaneh; Haron, Mohd Syamsul Reza; Tan, Sheau Wei; Omar, Abdul Rahman

    2015-11-01

    Apoptosis has been postulated to play an important role during feline infectious peritonitis virus (FIPV) infection; however, its mechanism is not well characterized. This study is focused on apoptosis and transcriptional profiling of FIPV-infected cells following in vitro infection of CRFK cells with FIPV 79-1146 WSU. Flow cytometry was used to determine mode of cell death in first 42 h post infection (hpi). FIPV infected cells underwent early apoptosis at 9 hpi (p < 0.05) followed by late apoptosis at 12 hpi (p < 0.05) and necrosis from 24 hpi (p < 0.05). Then, next generation sequencing was performed on 9 hpi and control uninfected cells by Illumina analyzer. An aggregate of 4546 genes (2229 down-regulated and 2317 up-regulated) from 17 cellular process, 11 molecular functions and 130 possible biological pathways were affected by FIPV. 131 genes from apoptosis cluster (80 down-regulated and 51 up-regulated) along with increase of apoptosis, p53, p38 MAPK, VEGF and chemokines/cytokines signaling pathways were probably involved in apoptosis process. Six of the de-regulated genes expression (RASSF1, BATF2, MAGEB16, PDCD5, TNFα and TRAF2) and TNFα protein concentration were analyzed by RT-qPCR and ELISA, respectively, at different time-points. Up-regulations of both pro-apoptotic (i.e. PDCD5) and anti-apoptotic (i.e. TRAF2) were detected from first hpi and continuing to deregulate during apoptosis process in the infected cells. PMID:26386572

  11. [Functional activity of lymphoblastoid cells infected by human adenovirus type 2 and Epstein-Barr virus].

    PubMed

    Povnitsa, O Iu; Diachenko, N S; Nosach, L N; Olevinskaia, Z M; Zhovnovataia, V L; Polishchuk, V N; Spivak, N Ia

    2005-01-01

    The paper deals with the influence of the adenovirus (Ad) and Epstein-Barr virus (EBV) on functional activity of lymphocytes, in particular, the production of alpha- and gamma-interferons, tumor necrosis factor (TNF) in conditions of mono- or double infection of B- and T-phenotype (CEM) lymphoblastoid cells. It is shown, that Ad, EBV or both viruses induce high enough levels of interferon on both lines of cells and in control epithelial cells. The lymphoblastoid cells infected by viruses deep ability to synthesize alpha- and gamma-interferons under the influence of the corresponding inducers (Newcastle disease virus and hemagglutinine). Nevertheless, the levels of their formation are not high. Rather high parameters of activity of the tumor necrosis factor (TNF) were revealed during a day in the initial B95-8 cells and superinfected Ad after the effect of LPS of E. coli. Their activity in CEM cells also did not depend on the infection type. PMID:16018208

  12. Antiviral Responses by Swine Primary Bronchoepithelial Cells Are Limited Compared to Human Bronchoepithelial Cells Following Influenza Virus Infection

    PubMed Central

    Hauser, Mary J.; Dlugolenski, Daniel; Culhane, Marie R.; Wentworth, David E.; Tompkins, S. Mark; Tripp, Ralph A.

    2013-01-01

    Swine generate reassortant influenza viruses because they can be simultaneously infected with avian and human influenza; however, the features that restrict influenza reassortment in swine and human hosts are not fully understood. Type I and III interferons (IFNs) act as the first line of defense against influenza virus infection of respiratory epithelium. To determine if human and swine have different capacities to mount an antiviral response the expression of IFN and IFN-stimulated genes (ISG) in normal human bronchial epithelial (NHBE) cells and normal swine bronchial epithelial (NSBE) cells was evaluated following infection with human (H3N2), swine (H1N1), and avian (H5N3, H5N2, H5N1) influenza A viruses. Expression of IFNλ and ISGs were substantially higher in NHBE cells compared to NSBE cells following H5 avian influenza virus infection compared to human or swine influenza virus infection. This effect was associated with reduced H5 avian influenza virus replication in human cells at late times post infection. Further, RIG-I expression was lower in NSBE cells compared to NHBE cells suggesting reduced virus sensing. Together, these studies identify key differences in the antiviral response between human and swine respiratory epithelium alluding to differences that may govern influenza reassortment. PMID:23875024

  13. Single cell mass cytometry reveals remodeling of human T cell phenotypes by varicella zoster virus.

    PubMed

    Sen, Nandini; Mukherjee, Gourab; Arvin, Ann M

    2015-11-15

    The recent application of mass cytometry (CyTOF) to biology provides a 'systems' approach to monitor concurrent changes in multiple host cell factors at the single cell level. We used CyTOF to evaluate T cells infected with varicella zoster virus (VZV) infection, documenting virus-mediated phenotypic and functional changes caused by this T cell tropic human herpesvirus. Here we summarize our findings using two complementary panels of antibodies against surface and intracellular signaling proteins to elucidate the consequences of VZV-mediated perturbations on the surface and in signaling networks of infected T cells. CyTOF data was analyzed by several statistical, analytical and visualization tools including hierarchical clustering, orthogonal scaling, SPADE, viSNE, and SLIDE. Data from the mass cytometry studies demonstrated that VZV infection led to 'remodeling' of the surface architecture of T cells, promoting skin trafficking phenotypes and associated with concomitant activation of T-cell receptor and PI3-kinase pathways. This method offers a novel approach for understanding viral interactions with differentiated host cells important for pathogenesis. PMID:26213183

  14. Visualization of the African swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent protein-p54 membrane protein chimera

    SciTech Connect

    Hernaez, Bruno . E-mail: hernaez@inia.es; Escribano, Jose M. . E-mail: escriban@inia.es; Alonso, Covadonga . E-mail: calonso@inia.es

    2006-06-20

    Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the p54 envelope protein fused to the enhanced green fluorescent protein (EGFP). The incorporation of the fusion protein into the virus particle was confirmed immunologically and it was determined that p54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.

  15. B and T cells are required for mouse mammary tumor virus spread within the mammary gland.

    PubMed

    Golovkina, T V; Dudley, J P; Ross, S R

    1998-09-01

    Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors. PMID:9725233

  16. 75 FR 54419 - Environmental Impact Statement: Queens County, NY

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-09-07

    ... Federal Highway Administration Environmental Impact Statement: Queens County, NY AGENCY: Federal Highway... Environmental Impact Statement (EIS) for the proposed Bridge Rehabilitation and Interchange Improvements Project... considered will not have a significant impact on the environment. To address these bridge conditions,...

  17. Queens become workers: pesticides alter caste differentiation in bees

    PubMed Central

    dos Santos, Charles F.; Acosta, André L.; Dorneles, Andressa L.; dos Santos, Patrick D. S.; Blochtein, Betina

    2016-01-01

    Bees are important for the world biodiversity and economy because they provide key pollination services in forests and crops. However, pesticide use in crops has adversely affected (decreased) queen production because of increased mortality among larvae. Here, we demonstrated that in vitro-reared queens of a neotropical social bee species (Plebeia droryana) also showed high larval mortality after exposure to an organophosphate pesticide (chlorpyrifos) via larval food. Moreover, most of the surviving larvae that were destined to develop into queens became workers more likely because they ate less food than expected without pesticide skewing thus caste differentiation in this bee species. This adverse effect has not been previously reported for any other social insects, such as honeybees or bumblebees. Queens are essential for breeding and colony growth. Therefore, if our data are applicable to other pantropical social bee species across the globe, it is likely that these bees are at a serious risk of failure to form new colonies. PMID:27530246

  18. Radar detection of drones responding to honeybee queen pheromone.

    PubMed

    Loper, G M; Wolf, W W; Taylor, O R

    1993-09-01

    The response of honey bee (Apis mellifera L.) drones to queen pheromone(s) (either natural from a mated queen, or synthetic from a lure) was recorded using an X-band, ground-based radar. The distribution of drones (insect targets on the radar screen) changed from a scattered distribution to a line concentration (downwind) when the pheromone was released. Displacement within the line concentration was toward the pheromone. This response was seen as far as 800±15 m downwind from a lure with 10 mg of synthetic 9-oxodec-trans-2-enoic acid (9-ODA) and as far as 420±15 m from a mated queen. These studies demonstrate that queen pheromone can be detected by drones at much greater distances than previously believed and illustrate how X-band radar may be used to establish the distances at which insects of similar or larger size respond to pheromones. PMID:24249369

  19. Queens become workers: pesticides alter caste differentiation in bees.

    PubMed

    Dos Santos, Charles F; Acosta, André L; Dorneles, Andressa L; Dos Santos, Patrick D S; Blochtein, Betina

    2016-01-01

    Bees are important for the world biodiversity and economy because they provide key pollination services in forests and crops. However, pesticide use in crops has adversely affected (decreased) queen production because of increased mortality among larvae. Here, we demonstrated that in vitro-reared queens of a neotropical social bee species (Plebeia droryana) also showed high larval mortality after exposure to an organophosphate pesticide (chlorpyrifos) via larval food. Moreover, most of the surviving larvae that were destined to develop into queens became workers more likely because they ate less food than expected without pesticide skewing thus caste differentiation in this bee species. This adverse effect has not been previously reported for any other social insects, such as honeybees or bumblebees. Queens are essential for breeding and colony growth. Therefore, if our data are applicable to other pantropical social bee species across the globe, it is likely that these bees are at a serious risk of failure to form new colonies. PMID:27530246

  20. A Critical Look at the Queen Bee Syndrome

    ERIC Educational Resources Information Center

    Berry, Jane; Kushner, Richard

    1975-01-01

    Discusses the popular "Queen Bee" stereotype of successful female executives, and concludes that the stereotype is too narrow in focus and fails to take into account complex psychological and experiential variables. (Author/EJT)

  1. Malignant transformation of hamster cells following infection with bovine herpesvirus (infectious bovine rhinotracheitis virus.

    PubMed

    Michalski, F; Hsiung, G D

    1975-03-01

    Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5% of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells. PMID:165538

  2. Variant Human T-cell Lymphotropic Virus Type 1c and Adult T-cell Leukemia, Australia

    PubMed Central

    Cassar, Olivier; Bardy, Peter; Kearney, Daniel; Gessain, Antoine

    2013-01-01

    Human T-cell lymphotropic virus type 1 is endemic to central Australia among Indigenous Australians. However, virologic and clinical aspects of infection remain poorly understood. No attempt has been made to control transmission to indigenous children. We report 3 fatal cases of adult T-cell leukemia/lymphoma caused by human T-cell lymphotropic virus type 1 Australo-Melanesian subtype c. PMID:24047544

  3. A fast track influenza virus vaccine produced in insect cells.

    PubMed

    Cox, Manon M J; Hashimoto, Yoshifumi

    2011-07-01

    The viral surface protein hemagglutinin (HA) has been recognized as a key antigen in the host response to influenza virus in both natural infection and vaccination because neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system can be used for the production of recombinant HA molecules and is suitable for influenza vaccine production where annual adjustment of the vaccine is required. This expression system is generally considered safe with minimal potential for growth of human pathogens. Extensive characterization of this novel cell substrate has been performed, none of which has revealed the presence of adventitious agents. Multiple clinical studies have demonstrated that the vaccine is safe, well-tolerated and immunogenic. The baculovirus-insect cell system could, therefore, be used for the expedited production of a safe and efficacious influenza vaccine. As a result, this technology should provide a fast track worldwide solution for newly emerging influenza strains or pandemic preparedness within a few years. PMID:21784229

  4. Maternal influence on the acceptance of virgin queens introduced into Africanized honey bee (Apis mellifera) colonies.

    PubMed

    Moretto, G; Guerra, J C V; Kalvelage, H; Espindola, E

    2004-01-01

    The oviposition potential of honey bee queens decreases with age, therefore it is important to replace old queens with younger ones on a periodic basis. However, queen replacement is problematic, especially in Africanized honey bee colonies, since many introduced queens are not accepted, and virgin queens are less easily accepted than are mated queens. We assessed the influence of genetic origin (queen mother) on the acceptance of queens, when they were introduced as virgins into Africanized honey bee colonies. For this purpose, 12 daughter queens from each of 11 mother queens with no degree of kinship among themselves were introduced. Introductions were made monthly, for 12 months, though the winter months of June and July were not included, as there is little brood and drones are rare in winter. There was some seasonal variation in the acceptance rates; generally there was greater acceptance in months with good honey flows. However, the acceptance of introduced queens was influenced by their origin. The rate of acceptance of daughter queens from the 11 different mother queens varied significantly, ranging from 33 to 75%. There appears to be a genetic influence of the mother queen on the introduced queen acceptance rate. PMID:15614734

  5. Influenza virus intracellular replication dynamics, release kinetics, and particle morphology during propagation in MDCK cells.

    PubMed

    Frensing, Timo; Kupke, Sascha Y; Bachmann, Mandy; Fritzsche, Susanne; Gallo-Ramirez, Lili E; Reichl, Udo

    2016-08-01

    Influenza viruses are respiratory pathogens and can cause severe disease. The best protection against influenza is provided by annual vaccination. These vaccines are produced in embryonated chicken eggs or using continuous animal cell lines. The latter processes are more flexible and scalable to meet the growing global demand. However, virus production in cell cultures is more expensive. Hence, further research is needed to make these processes more cost-effective and robust. We studied influenza virus replication dynamics to identify factors that limit the virus yield in adherent Madin-Darby canine kidney (MDCK) cells. The cell cycle stage of MDCK cells had no impact during early infection. Yet, our results showed that the influenza virus RNA synthesis levels out already 4 h post infection at a time when viral genome segments are exported from the nucleus. Nevertheless, virus release occurred at a constant rate in the following 16 h. Thereafter, the production of infectious viruses dramatically decreased, but cells continued to produce particles contributing to the hemagglutination (HA) titer. The majority of these particles from the late phase of infection were deformed or broken virus particles as well as large membranous structures decorated with viral surface proteins. These changes in particle characteristics and morphology need to be considered for the optimization of influenza virus production and vaccine purification steps. Moreover, our data suggest that in order to achieve higher cell-specific yields, a prolonged phase of viral RNA synthesis and/or a more efficient release of influenza virus particles is required. PMID:27129532

  6. Avian sarcoma and leukosis virus-receptor interactions: From classical genetics to novel insights into virus-cell membrane fusion

    SciTech Connect

    Barnard, R.J.O.; Elleder, D.; Young, J.A.T. . E-mail: jyoung@salk.edu

    2006-01-05

    For over 40 years, avian sarcoma and leukosis virus (ASLV)-receptor interactions have been employed as a useful model system to study the mechanism of retroviral entry into cells. Pioneering studies on this system focused upon the genetic basis of the differential susceptibilities of different lines of chickens to infection by distinct subgroups of ASLV. These studies led to the definition of three distinct autosomal recessive genes that were predicted to encode cellular receptors for different viral subgroups. They also led to the concept of viral interference, i.e. the mechanism by which infection by one virus can render cells resistant to reinfection by other viruses that use the same cellular receptor. Here, we review the contributions that analyses of the ASLV-receptor system have made in unraveling the mechanisms of retroviral entry into cells and focus on key findings such as identification and characterization of the ASLV receptor genes and the subsequent elucidation of an unprecedented mechanism of virus-cell fusion. Since many of the initial findings on this system were published in the early volumes of Virology, this subject is especially well suited to this special anniversary issue of the journal.

  7. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion

    SciTech Connect

    Simmons, Graham; Bertram, Stephanie; Glowacka, Ilona; Steffen, Imke; Chaipan, Chawaree; Agudelo, Juliet; Lu Kai; Rennekamp, Andrew J.; Hofmann, Heike; Bates, Paul; Poehlmann, Stefan

    2011-05-10

    Severe acute respiratory syndrome coronavirus (SARS-CoV) poses a considerable threat to human health. Activation of the viral spike (S)-protein by host cell proteases is essential for viral infectivity. However, the cleavage sites in SARS-S and the protease(s) activating SARS-S are incompletely defined. We found that R667 was dispensable for SARS-S-driven virus-cell fusion and for SARS-S-activation by trypsin and cathepsin L in a virus-virus fusion assay. Mutation T760R, which optimizes the minimal furin consensus motif 758-RXXR-762, and furin overexpression augmented SARS-S activity, but did not result in detectable SARS-S cleavage. Finally, SARS-S-driven cell-cell fusion was independent of cathepsin L, a protease essential for virus-cell fusion. Instead, a so far unknown leupeptin-sensitive host cell protease activated cellular SARS-S for fusion with target cells expressing high levels of ACE2. Thus, different host cell proteases activate SARS-S for virus-cell and cell-cell fusion and SARS-S cleavage at R667 and 758-RXXR-762 can be dispensable for SARS-S activation.

  8. Invariant NKT cells regulate the CD8 T cell response during Theiler's virus infection.

    PubMed

    Mars, Lennart T; Mas, Magali; Beaudoin, Lucie; Bauer, Jan; Leite-de-Moraes, Maria; Lehuen, Agnès; Bureau, Jean-Francois; Liblau, Roland S

    2014-01-01

    Invariant NKT cells are innate lymphocytes with a broad tissue distribution. Here we demonstrate that iNKT cells reside in the central nervous system (CNS) in the absence of inflammation. Their presence in the CNS dramatically augments following inoculation of C57Bl/6 mice with the neurotropic Theiler's murine encephalomyelitis virus (TMEV). At the peak of inflammation the cellular infiltrate comprises 45,000 iNKT cells for 1250 CD8 T cells specific for the immunodominant TMEV epitope. To study the interaction between these two T cell subsets, we infected both iNKT cell deficient Jα18(-/-) mice and iNKT cell enriched Vα14 transgenic mice with TMEV. The CD8 T cell response readily cleared TMEV infection in the iNKT cell deficient mice. However, in the iNKT cell enriched mice TMEV infection persisted and was associated with significant mortality. This was caused by the inhibition of the CD8 T cell response in the cervical lymph nodes and spleen after T cell priming. Taken together we demonstrate that iNKT cells reside in the CNS in the absence of inflammation and that their enrichment is associated with the inhibition of the anti-viral CD8 T cell response and an augmented mortality during acute encephalomyelitis. PMID:24498175

  9. Virus uncoating is required for apoptosis induction in cultured mammalian cells infected with African horse sickness virus.

    PubMed

    Vermaak, Elaine; Theron, Jacques

    2015-07-01

    Infection of cultured mammalian cells with African horse sickness virus (AHSV) is known to induce cell death. To date, the trigger(s) of this response, the apoptotic pathways activated during AHSV infection and the functional consequences of apoptosis on the virus replication cycle have yet to be characterized. This study demonstrated that extracellular treatment of BHK-21 cells with both of the AHSV4 outer capsid proteins, VP2 and VP5, was sufficient to trigger apoptosis. Whether steps in AHSV4 replication subsequent to viral attachment were required for AHSV4-induced apoptosis was also investigated. Apoptosis was induced in BHK-21 cells infected with UV-inactivated AHSV4 and in ribavirin-treated cells infected with AHSV4. However, both AHSV4- and VP2/VP5-stimulated apoptotic responses were inhibited in the presence of the endosomal acidification inhibitors ammonium chloride and chloroquine. These results indicated that uncoating of AHSV4 virions, but not viral transcription or subsequent steps in viral replication, was required for AHSV4 to induce apoptosis in BHK-21 cells. Furthermore, this study showed that both the extrinsic (caspase-8) and intrinsic (caspase-9) apoptotic pathways were induced following AHSV4 infection. The inhibition of caspase activity in AHSV4-infected cells did not diminish AHSV4 replication, but reduced the release and dissemination of progeny viral particles. Taken together, the data indicated that uncoating of AHSV virions was required for apoptosis induction, and that apoptosis enhanced virus spread and release. PMID:25783475

  10. Mitophagy switches cell death from apoptosis to necrosis in NSCLC cells treated with oncolytic measles virus.

    PubMed

    Xia, Mao; Meng, Gang; Jiang, Aiqin; Chen, Aiping; Dahlhaus, Meike; Gonzalez, Patrick; Beltinger, Christian; Wei, Jiwu

    2014-06-15

    Although apoptotic phenomena have been observed in malignant cells infected by measles virus vaccine strain Edmonston B (MV-Edm), the precise oncolytic mechanisms are poorly defined. In this study we found that MV-Edm induced autophagy and sequestosome 1-mediated mitophagy leading to decreased cytochrome c release, which blocked the pro-apoptotic cascade in non-small cell lung cancer cells (NSCLCs). The decrease of apoptosis by mitophagy favored viral replication. Persistent viral replication sustained by autophagy ultimately resulted in necrotic cell death due to ATP depletion. Importantly, when autophagy was impaired in NSCLCs MV-Edm-induced cell death was significantly abrogated despite of increased apoptosis. Taken together, our results define a novel oncolytic mechanism by which mitophagy switches cell death from apoptosis to more efficient necrosis in NSCLCs following MV-Edm infection. This provides a foundation for future improvement of oncolytic virotherapy or antiviral therapy. PMID:25004098

  11. Mating triggers dynamic immune regulations in wood ant queens.

    PubMed

    Castella, G; Christe, P; Chapuisat, M

    2009-03-01

    Mating can affect female immunity in multiple ways. On the one hand, the immune system may be activated by pathogens transmitted during mating, sperm and seminal proteins, or wounds inflicted by males. On the other hand, immune defences may also be down-regulated to reallocate resources to reproduction. Ants are interesting models to study post-mating immune regulation because queens mate early in life, store sperm for many years, and use it until their death many years later, while males typically die after mating. This long-term commitment between queens and their mates limits the opportunity for sexual conflict but raises the new constraint of long-term sperm survival. In this study, we examine experimentally the effect of mating on immunity in wood ant queens. Specifically, we compared the phenoloxidase and antibacterial activities of mated and virgin Formica paralugubris queens. Queens had reduced levels of active phenoloxidase after mating, but elevated antibacterial activity 7 days after mating. These results indicate that the process of mating, dealation and ovary activation triggers dynamic patterns of immune regulation in ant queens that probably reflect functional responses to mating and pathogen exposure that are independent of sexual conflict. PMID:19170815

  12. Unequal subfamily proportions among honey bee queen and worker brood

    PubMed

    Tilley; Oldroyd

    1997-12-01

    Queens from three colonies of feral honey bees, Apis mellifera were removed and placed in separate nucleus colonies. For each colony, eggs and larvae were taken from the nucleus and placed in the main hive on each of 3-4 consecutive weeks. Workers in the queenless parts selected young larvae to rear as queens. Queen pupae, together with the surrounding worker pupae, were removed from each colony and analysed at two to three microsatellite loci to determine their paternity. In all three colonies, the paternity of larvae chosen by the bees to rear as queens was not a random sample of the paternities in the worker brood, with certain subfamilies being over-represented in queens. These results support an important prediction of kin selection theory: when colonies are queenless, unequal relatedness within colonies could lead to the evolution of reproductive competition, that is some subfamilies achieving greater reproductive success than others. The mechanism by which such dominance is achieved could be through a system of kin recognition and nepotism, but we conclude that genetically based differential attractiveness of larvae for rearing as queens is more likely.Copyright 1997 The Association for the Study of Animal BehaviourCopyright 1997The Association for the Study of Animal Behaviour. PMID:9521799

  13. Chaotic Red Queen coevolution in three-species food chains

    PubMed Central

    Dercole, Fabio; Ferriere, Regis; Rinaldi, Sergio

    2010-01-01

    Coevolution between two antagonistic species follows the so-called ‘Red Queen dynamics’ when reciprocal selection results in an endless series of adaptation by one species and counteradaptation by the other. Red Queen dynamics are ‘genetically driven’ when selective sweeps involving new beneficial mutations result in perpetual oscillations of the coevolving traits on the slow evolutionary time scale. Mathematical models have shown that a prey and a predator can coevolve along a genetically driven Red Queen cycle. We found that embedding the prey–predator interaction into a three-species food chain that includes a coevolving superpredator often turns the genetically driven Red Queen cycle into chaos. A key condition is that the prey evolves fast enough. Red Queen chaos implies that the direction and strength of selection are intrinsically unpredictable beyond a short evolutionary time, with greatest evolutionary unpredictability in the superpredator. We hypothesize that genetically driven Red Queen chaos could explain why many natural populations are poised at the edge of ecological chaos. Over space, genetically driven chaos is expected to cause the evolutionary divergence of local populations, even under homogenizing environmental fluctuations, and thus to promote genetic diversity among ecological communities over long evolutionary time. PMID:20356888

  14. Asexual queen succession in the higher termite Embiratermes neotenicus

    PubMed Central

    Fougeyrollas, Romain; Dolejšová, Klára; Sillam-Dussès, David; Roy, Virginie; Poteaux, Chantal; Hanus, Robert; Roisin, Yves

    2015-01-01

    Asexual queen succession (AQS), in which workers, soldiers and dispersing reproductives are produced sexually while numerous non-dispersing queens arise through thelytokous parthenogenesis, has recently been described in three species of lower termites of the genus Reticulitermes. Here, we show that AQS is not an oddity restricted to a single genus of lower termites, but a more widespread strategy occurring also in the most advanced termite group, the higher termites (Termitidae). We analysed the genetic structure in 10 colonies of the Neotropical higher termite Embiratermes neotenicus (Syntermitinae) using five newly developed polymorphic microsatellite loci. The colonies contained one primary king accompanied either by a single primary queen or by up to almost 200 neotenic queens. While the workers, the soldiers and most future dispersing reproductives were produced sexually, the non-dispersing neotenic queens originated through thelytokous parthenogenesis of the founding primary queen. Surprisingly, the mode of thelytoky observed in E. neotenicus is most probably automixis with central fusion, contrasting with the automixis with terminal fusion documented in Reticulitermes. The occurrence of AQS based on different mechanisms of ploidy restoration raises the hypothesis of an independent evolutionary origin of this unique reproductive strategy in individual lineages of lower and higher termites. PMID:26019158

  15. Quantification of Hepatitis C Virus Cell-to-Cell Spread Using a Stochastic Modeling Approach

    PubMed Central

    Martin, Danyelle N.; Perelson, Alan S.; Dahari, Harel

    2015-01-01

    ABSTRACT It has been proposed that viral cell-to-cell transmission plays a role in establishing and maintaining chronic infections. Thus, understanding the mechanisms and kinetics of cell-to-cell spread is fundamental to elucidating the dynamics of infection and may provide insight into factors that determine chronicity. Because hepatitis C virus (HCV) spreads from cell to cell and has a chronicity rate of up to 80% in exposed individuals, we examined the dynamics of HCV cell-to-cell spread in vitro and quantified the effect of inhibiting individual host factors. Using a multidisciplinary approach, we performed HCV spread assays and assessed the appropriateness of different stochastic models for describing HCV focus expansion. To evaluate the effect of blocking specific host cell factors on HCV cell-to-cell transmission, assays were performed in the presence of blocking antibodies and/or small-molecule inhibitors targeting different cellular HCV entry factors. In all experiments, HCV-positive cells were identified by immunohistochemical staining and the number of HCV-positive cells per focus was assessed to determine focus size. We found that HCV focus expansion can best be explained by mathematical models assuming focus size-dependent growth. Consistent with previous reports suggesting that some factors impact HCV cell-to-cell spread to different extents, modeling results estimate a hierarchy of efficacies for blocking HCV cell-to-cell spread when targeting different host factors (e.g., CLDN1 > NPC1L1 > TfR1). This approach can be adapted to describe focus expansion dynamics under a variety of experimental conditions as a means to quantify cell-to-cell transmission and assess the impact of cellular factors, viral factors, and antivirals. IMPORTANCE The ability of viruses to efficiently spread by direct cell-to-cell transmission is thought to play an important role in the establishment and maintenance of viral persistence. As such, elucidating the dynamics of cell-to-cell

  16. Evaluation of cytokine gene expression after avian influenza virus infection in avian cell lines and primary cell cultures

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The innate immune responses elicited by avian influenza virus (AIV) infection has been studied by measuring cytokine gene expression by relative real time PCR (rRT-PCR) in vitro, using both cell lines and primary cell cultures. Continuous cell lines offer advantages over the use of primary cell cult...

  17. Bovine viral diarrhea virus infection alters global transcription profiles in bovine endothelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea viruses (BVDV) are significant pathogens of cattle worldwide. These viruses exist in both non-cytopathic and cytopathic biotypes. Non-cytopathic BVDV can establish persistent lifelong infections in cattle and are a frequent contaminant of biological reagents such as cell cultur...

  18. BVD virus infection alters global transcription profiles in bovine endothelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bovine viral diarrhea viruses (BVDV) are a significant pathogens of cattle worldwide. These viruses exist in both non-cytopathic or cytopathic biotypes. Non-cytopathic BVDV can establish persistent lifelong infections in cattle and are a frequent contaminant of biological reagents such as cell cul...

  19. Identification of gene biomarkers for respiratory synctial virus infection in a bronchical epithelial cell line

    EPA Science Inventory

    Abstract: Respiratory syncytial virus (RSV) infection involves complex virus-host interplay. In this study, we analyzed gene expression in RSV-infected BEAS-2B cells to discover novel signaling pathways and biomarkers. We hybridized RNAs from RSV- or vehicle-treated BEAS-2B to ...

  20. Virus-host interactions in persistently FMDV-infected cells derived from bovine pharynx

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Foot-and-mouth disease virus (FMDV) produces a disease in cattle characterized by vesicular lesions and a persistent infection with asymptomatic low-level production of virus. Here we describe the establishment of a persistently infected primary cell culture derived from bovine pharynx tissue (PBPT)...

  1. Influence of herpes simplex virus infection on benzo(a)pyrene metabolism in monkey kidney cells

    SciTech Connect

    Degenhardt, J.H.; Whitcomb, B.; Hall, M.R.

    1984-01-01

    Current research in our laboratory is designed to investigate the intracellular interactions of BP with oncogenic DNA viruses of animals and humans. In this study, our purpose was to determine whether BP is metabolized in herpes simplex virus type 2 (HSV-2) infected cells and whether HSV-2 infection affects intracellular levels of the aryl hydrocarbon hydroxylase system necessary for BP metabolism.

  2. The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro.

    PubMed

    Ohgimoto, S; Ohgimoto, K; Niewiesk, S; Klagge, I M; Pfeuffer, J; Johnston, I C; Schneider-Schaulies, J; Weidmann, A; ter Meulen, V; Schneider-Schaulies, S

    2001-08-01

    Recombinant measles viruses (MV) in which the authentic glycoprotein genes encoding the fusion and the haemagglutinin (H) proteins of the Edmonston (ED) vaccine strains were swapped singly or doubly for the corresponding genes of a lymphotropic MV wild-type virus (strain WTF) were used previously to investigate MV tropism in cell lines in tissue culture. When these recombinants and their parental strains, the molecular ED-based clone (ED-tag) and WTF, were used to infect cotton rats, only viruses expressing the MV WTF H protein replicated in secondary lymphatic tissues and caused significant immunosuppression. In vitro, viruses containing the ED H protein revealed a tropism for human peripheral blood lymphocytes as documented by enhanced binding and virus production, whereas those containing the WTF H protein replicated well in monocyte-derived dendritic cells (Mo-DC). This did not correlate with more efficient binding of these viruses to DC, but with an enhancement of uptake, virus spread, accumulation of viral antigens and virus production. Thus, replacement of the ED H protein with WTF H protein was sufficient to confer the DC tropism of WTF to ED-tag in vitro. This study suggests that the MV H protein plays an important role in determining cell tropism to immune cells and this may play an important role in the induction of immunosuppression in vivo. PMID:11457989

  3. AUTOGRAPHA CALIFORNICA NUCLEAR POLYHEDROSIS VIRUS EFFICIENTLY ENTERS BUT DOES NOT REPLICATE IN POIKILOTHERMIC VERTEBRATE CELLS

    EPA Science Inventory

    The host range of the insect virus Autographa californica nuclear polyhedrosis virus (AcMNPV) was examined. AcMNPV could not initiate a productive infection in frog, turtle, trout, or moth cell lines. After exposure to AcMNPV, neither viral DNA nor RNA synthesis could be detected...

  4. NK Cells during Dengue Disease and Their Recognition of Dengue Virus-Infected cells

    PubMed Central

    Beltrán, Davis; López-Vergès, Sandra

    2014-01-01

    The innate immune response, in addition to the B- and T-cell response, plays a role in protection against dengue virus (DENV) infection and the degree of disease severity. Early activation of natural killer (NK) cells and type-I interferon-dependent immunity may be important in limiting viral replication during the early stages of DENV infection and thus reducing subsequent pathogenesis. NK cells may also produce cytokines that reduce inflammation and tissue injury. On the other hand, NK cells are also capable of inducing liver injury at early-time points of DENV infection. In vitro, NK cells can kill antibody-coated DENV-infected cells through antibody-dependent cell-mediated cytotoxicity. In addition, NK cells may directly recognize DENV-infected cells through their activating receptors, although the increase in HLA class I expression may allow infected cells to escape the NK response. Recently, genome-wide association studies have shown an association between MICB and MICA, which encode ligands of the activating NK receptor NKG2D, and dengue disease outcome. This review focuses on recognition of DENV-infected cells by NK cells and on the regulation of expression of NK cell ligands by DENV. PMID:24829565

  5. NK Cells Help Induce Anti-Hepatitis B Virus CD8+ T Cell Immunity in Mice.

    PubMed

    Zheng, Meijuan; Sun, Rui; Wei, Haiming; Tian, Zhigang

    2016-05-15

    Although recent clinical studies demonstrate that NK cell function is impaired in hepatitis B virus (HBV)-persistent patients, whether or how NK cells play a role in anti-HBV adaptive immunity remains to be explored. Using a mouse model mimicking acute HBV infection by hydrodynamic injection of an HBV plasmid, we observed that although serum hepatitis B surface Ag and hepatitis B envelope Ag were eliminated within 3 to 4 wk, HBV might persist for >8 wk in CD8(-/-) mice and that adoptive transfer of anti-HBV CD8(+) T cells restored the ability to clear HBV in HBV-carrier Rag1(-/-) mice. These results indicate that CD8(+) T cells are critical in HBV elimination. Furthermore, NK cells increased IFN-γ production after HBV plasmid injection, and NK cell depletion led to significantly increased HBV persistence along with reduced frequency of hepatitis B core Ag-specific CD8(+) T cells. Adoptive transfer of IFN-γ-sufficient NK cells restored donor CD8(+) T cell function, indicating that NK cells positively regulated CD8(+) T cells via secreting IFN-γ. We also observed that NK cell depletion correlated with decreased effector memory CD8(+) T cell frequencies. Importantly, adoptive transfer experiments showed that NK cells were involved in anti-HBV CD8(+) T cell recall responses. Moreover, DX5(+)CD49a(-) conventional, but not DX5(-)CD49a(+) liver-resident, NK cells were involved in improving CD8(+) T cell responses against HBV. Overall, the current study reveals that NK cells, especially DX5(+)CD49a(-) conventional NK cells, promote the antiviral activity of CD8(+) T cell responses via secreting IFN-γ in a mouse model mimicking acute HBV infection. PMID:27183639

  6. Herpes simplex virus-mediated human hypoxanthine-guanine phosphoribosyltransferase gene transfer into neuronal cells

    SciTech Connect

    Palella, T.D.; Silverman, L.J.; Schroll, C.T.; Homa, F.L.; Levine, M.; Kelley, W.N.

    1988-01-01

    The virtually complete deficiency of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a devastating neurological disease, Lesch-Nyhan syndrome. Transfer of the HPRT gene into fibroblasts and lymphoblasts in vitro and into hematopoietic cells in vivo has been accomplished by other groups with retroviral-derived vectors. It appears to be necessary, however, to transfer the HPRT gene into neuronal cells to correct the neurological dysfunction of this disorder. The neurotropic virus herpes simplex virus type 1 has features that make it suitable for use as a vector to transfer the HPRT gene into neuronal tissue. This report describes the isolation of an HPRT-deficient rat neuroma cell line, designated B103-4C, and the construction of a recombinant herpes simplex virus type 1 that contained human HPRT cDNA. These recombinant viruses were used to infect B103-4C cells. Infected cells expressed HPRT activity which was human in origin.

  7. Human Respiratory Syncytial Virus Memphis 37 Grown in HEp-2 Cells Causes more Severe Disease in Lambs than Virus Grown in Vero Cells

    PubMed Central

    Derscheid, Rachel J.; van Geelen, Albert; McGill, Jodi L.; Gallup, Jack M.; Cihlar, Tomas; Sacco, Randy E.; Ackermann, Mark R.

    2013-01-01

    Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis in infants and young children. A small percentage of these individuals develop severe and even fatal disease. To better understand the pathogenesis of severe disease and develop therapies unique to the less-developed infant immune system, a model of infant disease is needed. The neonatal lamb pulmonary development and physiology is similar to that of infants, and sheep are susceptible to ovine, bovine, or human strains of RSV. RSV grown in Vero (African green monkey) cells has a truncated attachment G glycoprotein as compared to that grown in HEp-2 cells. We hypothesized that the virus grown in HEp-2 cells would cause more severe clinical symptoms and cause more severe pathology. To confirm the hypothesis, lambs were inoculated simultaneously by two different delivery methods (intranasal and nebulized inoculation) with either Vero-grown or HEp-2-grown RSV Memphis 37 (M37) strain of virus to compare viral infection and disease symptoms. Lambs infected with HEp-2 cell-derived virus by either intranasal or nebulization inoculation had significantly higher levels of viral RNA in lungs as well as greater clinical disease including both gross and histopathologic lesions compared to lambs similarly inoculated with Vero-grown virus. Thus, our results provide convincing in vivo evidence for differences in viral infectivity that corroborate previous in vitro mechanistic studies demonstrating differences in the G glycoprotein expression by RSV grown in Vero cells. PMID:24284879

  8. Multiplicity of virus-encoded helper T-cell epitopes expressed on FBL-3 tumor cells.

    PubMed Central

    Iwashiro, M; Kondo, T; Shimizu, T; Yamagishi, H; Takahashi, K; Matsubayashi, Y; Masuda, T; Otaka, A; Fujii, N; Ishimoto, A

    1993-01-01

    To identify retroviral antigenic determinants recognized by CD4+ T helper cells during tumor rejection, we established four noncytolytic, helper-type, CD4+ T-cell clones by limiting dilution cultures of mixed lymphocyte-tumor cultures from mice immune to a Friend virus-induced tumor, FBL-3. Among these, three T helper cell clones were isolated from C57BL/6 mice and the fourth was isolated from a (BALB/c x C57BL/6)F1 mouse. All these clones proliferated in response to the immunizing FBL-3 tumor cells in a major histocompatibility complex class II-restricted manner. Each clone expressed a distinct T-cell receptor with a characteristic combination of alpha and beta chains. The localization of helper T-cell determinants on viral proteins was analyzed with recombinant vaccinia viruses expressing Friend murine leukemia virus (F-MuLV) gag or env genes or shorter fragments of the env gene. Epitopes recognized by these T-cell clones were mapped to at least two distinct portions in the env region of the F-MuLV genome. These epitopes were identified more precisely with synthetic peptides derived from the F-MuLV envelope protein sequence. One of these epitopes was common to Friend and Moloney MuLVs and was located in the N-terminal region of the gp70 glycoprotein at amino acids 122 to 141. The second epitope, which was recognized in the context of hybrid I-Eb/d major histocompatibility complex class II molecule, was located close to the C-terminal end of gp70 at amino acids 462 to 479. In addition, a possible third epitope was located in the N-terminal half of the gp70 sequence and differed from the first epitope in that it was not cross-reactive with the Moloney MuLV envelope protein. PMID:7687300

  9. Generation of mink cell focus-forming viruses by Friend murine leukemia virus: recombination with specific endogenous proviral sequences.

    PubMed Central

    Evans, L H; Cloyd, M W

    1984-01-01

    A family of recombinant mink cell focus-forming viruses (MCF) was derived by inoculation of NFS mice with a Friend murine leukemia virus, and their genomes were analyzed by RNase T1-resistant oligonucleotide fingerprinting. The viruses were obtained from the thymuses and spleens of preleukemic and leukemic animals and were evaluated for dualtropism and oncogenicity. All these isolates induced cytopathic foci on mink cells but could be classified into two groups based on their relative infectivities for SC-1 (mouse) or mink (ATCC CCL64) cells. One group of Friend MCFs (F-MCFs) (group I) exhibited approximately equal infectivities for SC-1 and mink cells, whereas a second group (group II) infected mink cells 1,000- to 10,000-fold more efficiently than SC-1 cells. Structural analyses of the F-MCFs revealed that group I and group II viruses correlated with recombination of Friend murine leukemia virus with two distinct, but closely related, endogenous NFS proviral sequences. No correlation was found between the type of F-MCF and the tissue of origin or the disease state of the animal. Furthermore, none of the F-MCF isolates were found to be oncogenic in NFS/N or AKR/J mice. F-MCFs of both groups underwent extensive substitution of ecotropic sequences, involving much of the gag and env genes of group I F-MCFs and most of the gag, pol, and env genes of group II F-MCFs. All F-MCF isolates retained the 3' terminal U3 region of Friend murine leukemia virus. Comparison of the RNAs of the F-MCFs with RNAs of MCFs derived from NFS.Akv-1 or NFS.Akv-2 mice indicated that the F-MCFs were derived from NFS proviral sequences which are distinct from the sequences contained in NFS.Akv MCF isolates. This result suggested that recombination with particular endogenous proviral sequences to generate MCFs may be highly specific for a given murine leukemia virus. Images PMID:6422051

  10. Single-cell analysis and stochastic modelling unveil large cell-to-cell variability in influenza A virus infection

    PubMed Central

    Heldt, Frank S.; Kupke, Sascha Y.; Dorl, Sebastian; Reichl, Udo; Frensing, Timo

    2015-01-01

    Biochemical reactions are subject to stochastic fluctuations that can give rise to cell-to-cell variability. Yet, how this variability affects viral infections, which themselves involve noisy reactions, remains largely elusive. Here we present single-cell experiments and stochastic simulations that reveal a large heterogeneity between influenza A virus (IAV)-infected cells. In particular, experimental data show that progeny virus titres range from 1 to 970 plaque-forming units and intracellular viral RNA (vRNA) levels span three orders of magnitude. Moreover, the segmentation of IAV genomes seems to increase the susceptibility of their replication to noise, since the level of different genome segments can vary substantially within a cell. In addition, simulations suggest that the abortion of virus entry and random degradation of vRNAs can result in a large fraction of non-productive cells after single-hit infection. These results challenge current beliefs that cell population measurements and deterministic simulations are an accurate representation of viral infections. PMID:26586423

  11. The Role of B Cells for in Vivo T Cell Responses to a Friend Virus-Induced Leukemia

    NASA Astrophysics Data System (ADS)

    Schultz, Kirk R.; Klarnet, Jay P.; Gieni, Randall S.; Hayglass, Kent T.; Greenberg, Philip D.

    1990-08-01

    B cells can function as antigen-presenting cells and accessory cells for T cell responses. This study evaluated the role of B cells in the induction of protective T cell immunity to a Friend murine leukemia virus (F-MuLV)-induced leukemia (FBL). B cell-deficient mice exhibited significantly reduced tumor-specific CD4^+ helper and CD8^+ cytotoxic T cell responses after priming with FBL or a recombinant vaccinia virus containing F-MuLV antigens. Moreover, these mice had diminished T cell responses to the vaccinia viral antigens. Tumor-primed T cells transferred into B cell-deficient mice effectively eradicated disseminated FBL. Thus, B cells appear necessary for efficient priming but not expression of tumor and viral T cell immunity.

  12. Shared alterations in NK cell frequency, phenotype, and function in chronic human immunodeficiency virus and hepatitis C virus infections.

    PubMed

    Meier, Ute-Christiane; Owen, Rachel E; Taylor, Elizabeth; Worth, Andrew; Naoumov, Nikolai; Willberg, Christian; Tang, Kwok; Newton, Phillipa; Pellegrino, Pierre; Williams, Ian; Klenerman, Paul; Borrow, Persephone

    2005-10-01

    Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) cause clinically important persistent infections. The effects of virus persistence on innate immunity, including NK cell responses, and the underlying mechanisms are not fully understood. We examined the frequency, phenotype, and function of peripheral blood CD3- CD56+ NK subsets in HIV+ and HCV+ patients and identified significantly reduced numbers of total NK cells and a striking shift in NK subsets, with a marked decrease in the CD56(dim) cell fraction compared to CD56(bright) cells, in both infections. This shift influenced the phenotype and functional capacity (gamma interferon production, killing) of the total NK pool. In addition, abnormalities in the functional capacity of the CD56(dim) NK subset were observed in HIV+ patients. The shared NK alterations were found to be associated with a significant reduction in serum levels of the innate cytokine interleukin 15 (IL-15). In vitro stimulation with IL-15 rescued NK cells of HIV+ and HCV+ patients from apoptosis and enhanced proliferation and functional activity. We hypothesize that the reduced levels of IL-15 present in the serum during HIV and HCV infections might impact NK cell homeostasis, contributing to the common alterations of the NK pool observed in these unrelated infections. PMID:16160163

  13. [Biochemical characteristics of a calf leukemia virus in chronically infected cells].

    PubMed

    Argirova, R

    1979-01-01

    Studied were the conditions of cultivation of FLK cells chronically infected with a calf leucosis virus. The gradient values of density were compared to those of the murine sarcoma virus--1.14--1.15 vs, 1.17--1.18/cm3. Established were the parameters of the reverse transcriptase reaction for the calf leukosis virus (Magnesium-dependent reverse transcriptase). Data showed that the calf leucosis virus may not resolutely be referred either to the B- or the the C-type of retroviruses. PMID:92095

  14. Transcriptomic response to injury sheds light on the physiological costs of reproduction in ant queens.

    PubMed

    von Wyschetzki, Katharina; Lowack, Helena; Heinze, Jürgen

    2016-05-01

    The trade-off between reproduction and longevity is widespread among multicellular organisms. As an important exception, the reproductive females of perennial social insects (ants, honeybees, termites) are simultaneously highly fertile and very long-lived relative to their nonreproductive nestmates. The observation that increased fecundity is not coupled with decreased lifespan suggests that social insect queens do not have to reallocate resources between reproduction and self-maintenance. If queens have to compensate for the costs of reproduction on the level of the individual, the activation of other energy-demanding physiological processes might force them to reduce the production of eggs. To test this hypothesis in ant queens, we increased immunity costs by injury and measured the effect of this treatment on egg-laying rates and genomewide gene expression. Amputation of both middle legs led to a temporary decrease in egg-laying rates and affected the expression of 947 genes corresponding to 9% of the transcriptome. The changes comprised the upregulation of the immune and wound healing response on the one hand, and the downregulation of germ cell development, central nervous system development and learning ability on the other hand. Injury strongly influenced metabolism by inducing catabolism and repressing amino acid and nitrogen compound metabolism. By comparing our results to similar transcriptomic studies in insects, we found a highly consistent upregulation of immune genes due to sterile and septic wounding. The gene expression changes, complemented by the temporary decline of egg-laying rates, clearly reveal a trade-off between reproduction and the immune response in social insect queens. PMID:26880273

  15. Adeno-associated virus sensitizes HeLa cell tumors to gamma rays.

    PubMed Central

    Walz, C; Schlehofer, J R; Flentje, M; Rudat, V; zur Hausen, H

    1992-01-01

    Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment. Images PMID:1323717

  16. [Testing the susceptibility of cultured cells to infection with bovine leukemia virus].

    PubMed

    Bobáková, M; Lesník, F; Vrtiak, O J

    1985-05-01

    Different cell cultures were studied for their susceptibility to bovine leucosis virus infection. Syncytial assay was used for this study. The FLS/BLV+ cell line served as virus source. Cell lines BHK-21 and ZP-1/58 were found to be susceptible to syncytium formation. Large cells with one to three large nuclei, and loose nuclei reaching the size of syncytium were observed to occur in the BHK-21 and ZP-1/58 cell lines, apart from the syncytial formations. The virus specificity of the syncytia arising in these two cell lines was confirmed by the immunofluorescence assay. In the case of the immunoperoxidase assay, a positive result was obtained only in the BHK-21 cell line. The occurrence of syncytia and large nuclei was observed even in the cases when the BHK-21 cells were infected with the lymphocytes of leucotic cows. PMID:2992148

  17. Identification of morphological differences between avian influenza A viruses grown in chicken and duck cells.

    PubMed

    Al-Mubarak, Firas; Daly, Janet; Christie, Denise; Fountain, Donna; Dunham, Stephen P

    2015-03-01

    Although wild ducks are considered to be the major reservoirs for most influenza A virus subtypes, they are typically resistant to the effects of the infection. In contrast, certain influenza viruses may be highly pathogenic in other avian hosts such as chickens and turkeys, causing severe illness and death. Following in vitro infection of chicken and duck embryo fibroblasts (CEF and DEF) with low pathogenic avian influenza (LPAI) viruses, duck cells die more rapidly and produce fewer infectious virions than chicken cells. In the current study, the morphology of viruses produced from CEF and DEF cells infected with low pathogenic avian H2N3 was examined. Transmission electron microscopy showed that viruses budding from duck cells were elongated, while chicken cells produced mostly spherical virions; similar differences were observed in viral supernatants. Sequencing of the influenza genome of chicken- and duck-derived H2N3 LPAI revealed no differences, implicating host cell determinants as responsible for differences in virus morphology. Both DEF and CEF cells produced filamentous virions of equine H3N8 (where virus morphology is determined by the matrix gene). DEF cells produced filamentous or short filament virions of equine H3N8 and avian H2N3, respectively, even after actin disruption with cytochalasin D. These findings suggest that cellular factors other than actin are responsible for the formation of filamentous virions in DEF cells. The formation of elongated virions in duck cells may account for the reduced number of infectious virions produced and could have implications for virus transmission or maintenance in the reservoir host. PMID:25613009

  18. Quantitative analysis of virus and plasmid trafficking in cells

    NASA Astrophysics Data System (ADS)

    Lagache, Thibault; Dauty, Emmanuel; Holcman, David

    2009-01-01

    Intracellular transport of DNA carriers is a fundamental step of gene delivery. By combining both theoretical and numerical approaches we study here single and several viruses and DNA particles trafficking in the cell cytoplasm to a small nuclear pore. We present a physical model to account for certain aspects of cellular organization, starting with the observation that a viral trajectory consists of epochs of pure diffusion and epochs of active transport along microtubules. We define a general degradation rate to describe the limitations of the delivery of plasmid or viral particles to a nuclear pore imposed by various types of direct and indirect hydrolysis activity inside the cytoplasm. By replacing the switching dynamics by a single steady state stochastic description, we obtain estimates for the probability and the mean time for the first one of many particles to go from the cell membrane to a small nuclear pore. Computational simulations confirm that our model can be used to analyze and interpret viral trajectories and estimate quantitatively the success of nuclear delivery.

  19. Screening for Recombinant Avian Leukosis Viruses in Cell Cultures Inoculated with Various Subgroups of Virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chicken embryo fibroblasts (CEFs) prepared from ADOL SPF embryos were co-infected with different concentration ratios of subgroups A, J and E avian leukosis virus (ALV). Inoculated cultures were screened for recombination among the ALV strains. Potential recombinant viruses were purified by limiting...

  20. Eddies off the Queen Charlotte Islands

    NASA Technical Reports Server (NTRS)

    2002-01-01

    The bright red, green, and turquoise patches to the west of British Columbia's Queen Charlotte Islands and Alaska's Alexander Archipelago highlight the presence of biological activity in the ocean. These colors indicate high concentrations of chlorophyll, the primary pigment found in phytoplankton. Notice that there are a number of eddies visible in the Pacific Ocean in this pseudo-color scene. The eddies are formed by strong outflow currents from rivers along North America's west coast that are rich in nutrients from the springtime snowmelt running off the mountains. This nutrient-rich water helps stimulate the phytoplankton blooms within the eddies. (For more details, read Tracking Eddies that Feed the Sea.) To the west of the eddies in the water, another type of eddy-this one in the atmosphere-forms the clouds into the counterclockwise spiral characteristic of a low pressure system in the Northern Hemisphere. (Click on the image above to see it at full resolution; or click to see the scene in true-color.) The snow-covered mountains of British Columbia are visible in the upper righthand corner of the image. This scene was constructed using SeaWiFS data collected on June 13, 2002. SeaWiFS image courtesy the SeaWiFS Project, NASA/Goddard Space Flight Center, and ORBIMAGE

  1. Temperature-sensitive tumorigenicity of cells transformed by a mutant of Moloney sarcoma virus.

    PubMed Central

    Klarlund, J K; Forchhammer, J

    1980-01-01

    Normal rat kidney cells were nonproductively infected either with CP27, a mutant of Moloney sarcoma virus that is temperature-sensitive for maintenance of transformation, or with the parental wild-type virus. The nonproducer cells were inoculated into the tails of athymic nude mice that were subsequently incubated at 28 or 36 degrees C. CP27-infected cells induced tumors only at 28 degrees C, whereas cells infected with wild-type Moloney sarcoma virus were tumorigenic at both temperatures. Tumors induced at 28 degrees C by wild-type virus-infected cells grew faster after shift of the mice to 36 degrees C. In contrast, tumors induced by CP27-infected cells regressed upon shift to 36 degrees C, indicating that continuous expression of viral functions is required for persistence and growth of the tumors. After regression, secondary tumor growth was observed late after upshift of temperature-sensitive tumors. Cells recovered from these late-appearing tumors were tumorigenic at the nonpermissive temperature, and tumors induced by these cells did not regress after upshift. Virus rescued from these recovered cells retained the temperature-sensitivity for focus formation, indicating that the occurrence of the phenotypically wild-type cells was due to host cell modifications rather than to reversion of the CP27 genome. Images PMID:6929500

  2. Ebola virus VP40 late domains are not essential for viral replication in cell culture.

    PubMed

    Neumann, Gabriele; Ebihara, Hideki; Takada, Ayato; Noda, Takeshi; Kobasa, Darwyn; Jasenosky, Luke D; Watanabe, Shinji; Kim, Jin H; Feldmann, Heinz; Kawaoka, Yoshihiro

    2005-08-01

    Ebola virus particle formation and budding are mediated by the VP40 protein, which possesses overlapping PTAP and PPXY late domain motifs (7-PTAPPXY-13). These late domain motifs have also been found in the Gag proteins of retroviruses and the matrix proteins of rhabdo- and arenaviruses. While in vitro studies suggest a critical role for late domain motifs in the budding of these viruses, including Ebola virus, it remains unclear as to whether the VP40 late domains play a role in Ebola virus replication. Alteration of both late domain motifs drastically reduced VP40 particle formation in vitro. However, using reverse genetics, we were able to generate recombinant Ebola virus containing mutations in either or both of the late domains. Viruses containing mutations in one or both of their late domain motifs were attenuated by one log unit. Transmission and scanning electron microscopy did not reveal appreciable differences between the mutant and wild-type viruses released from infected cells. These findings indicate that the Ebola VP40 late domain motifs enhance virus replication but are not absolutely required for virus replication in cell culture. PMID:16051823

  3. Human immunodeficiency virus type 1 Tat protein modulates cell cycle and apoptosis in Epstein-Barr virus-immortalized B cells.

    PubMed

    Colombrino, Eva; Rossi, Elisabetta; Ballon, Gianna; Terrin, Liliana; Indraccolo, Stefano; Chieco-Bianchi, Luigi; De Rossi, Anita

    2004-05-01

    Patients infected with human immunodeficiency virus type 1 (HIV-1) develop a spectrum of B cell lymphoproliferative disorders ranging from polyclonal B cell activation to B cell lymphomas. While a direct role of Epstein-Barr virus (EBV) is well recognized for most of these lesions, recent findings have suggested that transactivator HIV-1 Tat protein might be involved in the pathogenesis of B cell lymphomas. Tat-expressing EBV-positive B cells were generated by transduction with a retroviral Tat-encoding vector. B(Tat+) cells expressed lower levels of anti-apoptotic protein Bcl-2 than parental and control B(Tat-) cells, generated by transduction with an empty retroviral vector, and were more prone to apoptosis upon serum withdrawal, as assessed by analysis of annexin V-stained cells and cleavage of poly-ADP-ribose-polymerase by caspase 3. Nevertheless, in serum starvation, B(Tat-) cells mainly exhibited the Rb hypo-phosphorylated form, underwent cell cycle arrest, and grew in single cell suspension, while B(Tat+) cells displayed the Rb hyper-phoshorylated form, progressed throughout the cell cycle, and retained the ability to grow in small clumps. Finding that B(Tat+) cells maintained proliferative capacity upon serum withdrawal suggests that cells expressing Tat have growth advantages among the EBV-driven cell proliferations and may originate B cell clones with more oncogenic potential. PMID:15093750

  4. Contact-Inhibited Revertant Cell Lines Isolated from Simian Virus 40-Transformed Cells III. Concanavalin A-Selected Revertant Cells

    PubMed Central

    Culp, Lloyd A.; Black, Paul H.

    1972-01-01

    Contact-inhibited variants have been isolated by treatment of simian virus 40 (SV40)-transformed Balb/c 3T3 cells (SVT2) with the plant lectin concanavalin A. These con A revertant cells exhibit the following properties: (i) they resemble 3T3 cells morphologically and grow to saturation densities which are similar to that of 3T3 cells; (ii) they synthesize the SV40-specific T antigen and yield infectious virus after fusion with permissive monkey cells; (iii) they contain a high sialic acid content similar to that of 3T3 cells and not to that of SVT2 cells; sialic acid composition was found to be independent of serum concentration; (iv) they contain more chromosomes with the average number in the tetraploid range than the SVT2 cells from which they were derived; and (v) SVT2 and revertant cells, confluent or subconfluent, produce more collagen than Balb/3T3 cells. The relationship of surface membrane properties to contact inhibition of growth and the mechanisms for generating revertant cells are discussed. Images PMID:4336561

  5. Multifunctional queen pheromone and maintenance of reproductive harmony in termite colonies.

    PubMed

    Matsuura, Kenji

    2012-06-01

    Pheromones are likely involved in all social activities of social insects including foraging, sexual behavior, defense, nestmate recognition, and caste regulation. Regulation of the number of fertile queens requires communication between reproductive and non-reproductive individuals. Queen-produced pheromones have long been believed to be the main factor inhibiting the differentiation of new reproductive individuals. However, since the discovery more than 50 years ago of the queen honeybee substance that inhibits the queen-rearing behavior of workers, little progress has been made in the chemical identification of inhibitory queen pheromones in other social insects. The recent identification of a termite queen pheromone and subsequent studies have elucidated the multifaceted roles of volatile pheromones, including functions such as a fertility signal, worker attractant, queen-queen communication signal, and antimicrobial agent. The proximate origin and evolutionary parsimony of the termite queen pheromone also are discussed. PMID:22623152

  6. Virgin honeybee queens fail to suppress worker fertility but not fertility signalling.

    PubMed

    Orlova, Margarita; Malka, Osnat; Hefetz, Abraham

    2013-03-01

    Queen mating status in social insects is a matter of crucial importance for workers because of its influence on the queen's productivity and consequently their fitness. Behavioural and physiological reactions of workers to the queens mating status have been studied as a proxy to mechanisms maintaining insect sociality. Here we show that unmated honeybee queens have considerably impaired capacity to trigger worker sterility and cooperative behaviour in comparison to mated (and thus more productive) queens and that under unmated queens social harmony in honeybee societies and queen's dominant position are somewhat compromised. Together with this it is shown that honeybee workers exposed to unmated queens despite being active reproductively and behaving accordingly display an impaired ability to advertise their fertility compared to queenless workers. These findings suggest that reproductive development, behavioural reactions and production of fertility signals are differentially regulated and differently influenced by the queen's presence. PMID:23232436

  7. Active influenza virus neuraminidase is expressed in monkey cells from cDNA cloned in simian virus 40 vectors.

    PubMed Central

    Davis, A R; Bos, T J; Nayak, D P

    1983-01-01

    We have replaced the late genes of simian virus 40 (SV40) with a cloned cDNA copy of the neuraminidase (NA; EC 3.2.1.18) gene of the WSN (H1N1) strain of human influenza virus. When the SV40-NA recombinant virus was complemented in a lytic infection of monkey cells with a helper virus containing an early region deletion mutant, influenza NA was expressed and readily detected by immunofluorescence as well as by immunoprecipitation of in vivo labeled proteins with monoclonal antibodies against NA. In addition, the expressed NA exhibited enzymatic activity by cleaving the sialic acid residue from alpha-2,3-sialyllactitol. The expressed protein was glycosylated and transported to the cell surface, and it possessed the same molecular weight as the NA of WSN virus grown in monkey cells. Because the structure of NA is quite different from that of other integral membrane proteins and includes an anchoring region at the NH2 terminus consisting of hydrophobic amino acids, we also constructed deletion mutants of NA in this region. Replacement of DNA coding for the first 10 NH2-terminal amino acids with SV40 and linker sequences had no apparent effect on NA expression, glycosylation, transport to the cell surface, or enzymatic activity. However, further deletion of NA DNA encoding the first 26 amino acids abolished NA expression. These data suggest that the hydrophobic NH2-terminal region is multifunctional and is important in biosynthesis and translocation of NA across the membrane as well as in anchoring the protein. Images PMID:6306656

  8. Viral Load Analysis of Hepatitis C Virus in Huh7.5 Cell Culture System

    PubMed Central

    Teimourpour, Roghayeh; Meshkat, Zahra; Gholoubi, Aida; Nomani, Hosein; Rostami, Sina

    2015-01-01

    Background: Previous studies using cell culture systems for the replication of hepatitis C virus have opened new research dimensions, and paved the ways for further and detailed studies of the virus in vitro. Objectives: The purpose of the present study was to cultivate hepatitis C virus in a cell culture system and evaluate viral amplification. Materials and Methods: In order to propagate hepatitis C virus, cloned whole genome of virus, JFH-1, was used. JFH-1 cDNA was introduced into strain JM109 of Escherichia coli and plasmid, containing the viral genome was purified from transformed bacteria. After XbaI digestion, RNA synthesis was induced using T7 RNA polymerase enzyme. Next, eukaryotic cell line Huh 7.5 was transfected by the purified RNA. Finally, Huh-7.5 cell line was infected with replicated virus and viral load was determined using real-time PCR (Polymerase Chain Reaction). Results: The amount of viral load, which was measured using real-time PCR was 17592 IU/mL. Conclusions: In the present study, using cell culture, a high titer (in acceptable range) of infectious hepatitis C virus was produced. This method could be used in future studies. PMID:26290686

  9. The ex vivo purge of cancer cells using oncolytic viruses: recent advances and clinical implications

    PubMed Central

    Tsang, Jovian J; Atkins, Harold L

    2015-01-01

    Hematological malignancies are treated with intensive high-dose chemotherapy, with or without radiation. This is followed by hematopoietic stem cell (HSC) transplantation (HSCT) to rescue or reconstitute hematopoiesis damaged by the anticancer therapy. Autologous HSC grafts may contain cancer cells and purging could further improve treatment outcomes. Similarly, allogeneic HSCT may be improved by selectively purging alloreactive effector cells from the graft rather than wholesale immune cell depletion. Viral agents that selectively replicate in specific cell populations are being studied in experimental models of cancer and immunological diseases and have potential applications in the context of HSC graft engineering. This review describes preclinical studies involving oncolytic virus strains of adenovirus, herpes simplex virus type 1, myxoma virus, and reovirus as ex vivo purging agents for HSC grafts, as well as in vitro and in vivo experimental studies using oncolytic coxsackievirus, measles virus, parvovirus, vaccinia virus, and vesicular stomatitis virus to eradicate hematopoietic malignancies. Alternative ex vivo oncolytic virus strategies are also outlined that aim to reduce the risk of relapse following autologous HSCT and mitigate morbidity and mortality due to graft-versus-host disease in allogeneic HSCT. PMID:27512666

  10. Herpes B Virus, Macacine Herpesvirus 1, Breaks Simplex Virus Tradition via Major Histocompatibility Complex Class I Expression in Cells from Human and Macaque Hosts

    PubMed Central

    Vasireddi, Mugdha

    2012-01-01

    B virus of the family Herpesviridae is endemic to rhesus macaques but results in 80% fatality in untreated humans who are zoonotically infected. Downregulation of major histocompatibility complex (MHC) class I in order to evade CD8+ T-cell activation is characteristic of most herpesviruses. Here we examined the cell surface presence and total protein expression of MHC class I molecules in B virus-infected human foreskin fibroblast cells and macaque kidney epithelial cells in culture, which are representative of foreign and natural host initial target cells of B virus. Our results show <20% downregulation of surface MHC class I molecules in either type of host cells infected with B virus, which is statistically insignificantly different from that observed in uninfected cells. We also examined the surface expression of MHC class Ib molecules, HLA-E and HLA-G, involved in NK cell inhibition. Our results showed significant upregulation of HLA-E and HLA-G in host cells infected with B virus relative to the amounts observed in other herpesvirus-infected cells. These results suggest that B virus-infected cell surfaces maintain normal levels of MHC class Ia molecules, a finding unique among simplex viruses. This is a unique divergence in immune evasion for B virus, which, unlike human simplex viruses, does not inhibit the transport of peptides for loading onto MHC class Ia molecules because B virus ICP47 lacks a transporter-associated protein binding domain. The fact that MHC class Ib molecules were significantly upregulated has additional implications for host-pathogen interactions. PMID:22973043

  11. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs

    PubMed Central

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  12. Sterilizing immunity to influenza virus infection requires local antigen-specific T cell response in the lungs.

    PubMed

    Dutta, Avijit; Huang, Ching-Tai; Lin, Chun-Yen; Chen, Tse-Ching; Lin, Yung-Chang; Chang, Chia-Shiang; He, Yueh-Chia

    2016-01-01

    Sterilizing immunity is a unique immune status, which prevents effective virus infection into the host. It is different from the immunity that allows infection but with subsequent successful eradication of the virus. Pre-infection induces sterilizing immunity to homologous influenza virus challenge in ferret. In our antigen-specific experimental system, mice pre-infected with PR8 influenza virus through nasal route are likewise resistant to reinfection of the same strain of virus. The virus is cleared before establishment of effective infection. Intramuscular influenza virus injection confers protection against re-infection with facilitated virus clearance but not sterilizing immunity. Pre-infection and intramuscular injection generates comparable innate immunity and antibody response, but only pre-infection induces virus receptor reduction and efficient antigen-specific T cell response in the lungs. Pre-infection with nH1N1 influenza virus induces virus receptor reduction but not PR8-specific T cell immune response in the lungs and cannot prevent infection of PR8 influenza virus. Pre-infection with PR8 virus induced PR8-specific T cell response in the lungs but cannot prevent infection of nH1N1 virus either. These results reveal that antigen-specific T cell immunity is required for sterilizing immunity. PMID:27596047

  13. Over-expression of putative transcriptional coactivator KELP interferes with Tomato mosaic virus cell-to-cell movement.

    PubMed

    Sasaki, Nobumitsu; Ogata, Takuya; Deguchi, Masakazu; Nagai, Shoko; Tamai, Atsushi; Meshi, Tetsuo; Kawakami, Shigeki; Watanabe, Yuichiro; Matsushita, Yasuhiko; Nyunoya, Hiroshi

    2009-03-01

    Tomato mosaic virus (ToMV) encodes a movement protein (MP) that is necessary for virus cell-to-cell movement. We have demonstrated previously that KELP, a putative transcriptional coactivator of Arabidopsis thaliana, and its orthologue from Brassica campestris can bind to ToMV MP in vitro. In this study, we examined the effects of the transient over-expression of KELP on ToMV infection and the intracellular localization of MP in Nicotiana benthamiana, an experimental host of the virus. In co-bombardment experiments, the over-expression of KELP inhibited virus cell-to-cell movement. The N-terminal half of KELP (KELPdC), which had been shown to bind to MP, was sufficient for inhibition. Furthermore, the over-expression of KELP and KELPdC, both of which were co-localized with ToMV MP, led to a reduction in the plasmodesmal association of MP. In the absence of MP expression, KELP was localized in the nucleus and the cytoplasm by the localization signal in its N-terminal half. It was also shown that ToMV amplified normally in protoplasts prepared from leaf tissue that expressed KELP transiently. These results indicate that over-expressed KELP interacts with MP in vivo and exerts an inhibitory effect on MP function for virus cell-to-cell movement, but not on virus amplification in individual cells. PMID:19236566

  14. Deformed wing virus implicated in overwintering honeybee colony losses.

    PubMed

    Highfield, Andrea C; El Nagar, Aliya; Mackinder, Luke C M; Noël, Laure M-L J; Hall, Matthew J; Martin, Stephen J; Schroeder, Declan C

    2009-11-01

    The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses. PMID:19783750

  15. Deformed Wing Virus Implicated in Overwintering Honeybee Colony Losses ▿

    PubMed Central

    Highfield, Andrea C.; El Nagar, Aliya; Mackinder, Luke C. M.; Noël, Laure M.-L. J.; Hall, Matthew J.; Martin, Stephen J.; Schroeder, Declan C.

    2009-01-01

    The worldwide decline in honeybee colonies during the past 50 years has often been linked to the spread of the parasitic mite Varroa destructor and its interaction with certain honeybee viruses. Recently in the United States, dramatic honeybee losses (colony collapse disorder) have been reported; however, there remains no clear explanation for these colony losses, with parasitic mites, viruses, bacteria, and fungal diseases all being proposed as possible candidates. Common characteristics that most failing colonies share is a lack of overt disease symptoms and the disappearance of workers from what appears to be normally functioning colonies. In this study, we used quantitative PCR to monitor the presence of three honeybee viruses, deformed wing virus (DWV), acute bee paralysis virus (ABPV), and black queen cell virus (BQCV), during a 1-year period in 15 asymptomatic, varroa mite-positive honeybee colonies in Southern England, and 3 asymptomatic colonies confirmed to be varroa mite free. All colonies with varroa mites underwent control treatments to ensure that mite populations remained low throughout the study. Despite this, multiple virus infections were detected, yet a significant correlation was observed only between DWV viral load and overwintering colony losses. The long-held view has been that DWV is relatively harmless to the overall health status of honeybee colonies unless it is in association with severe varroa mite infestations. Our findings suggest that DWV can potentially act independently of varroa mites to bring about colony losses. Therefore, DWV may be a major factor in overwintering colony losses. PMID:19783750

  16. Mantle cell lymphoma salvage regimen: synergy between a reprogrammed oncolytic virus and two chemotherapeutics

    PubMed Central

    Ungerechts, Guy; Frenzke, Marie E; Yaiw, Koon-Chu; Miest, Tanner; Johnston, Patrick B; Cattaneo, Roberto

    2010-01-01

    MV-PNP HblindantiCD20 is a CD20-targeted and prodrug convertase-armed measles virus (MV) that temporarily controls growth of lymphoma xenografts in SCID mice in combination with fludarabine phosphate. Herein, we examine the replication of this targeted virus and of a vaccine-lineage MV in disease bulks and circulating cells from mantle cell lymphoma (MCL) patients, and show that only the targeted virus is specific for CD20-expressing cells. We then assessed the efficacy of different regimens of administration of this virus in combination with fludarabine and cyclophosphamide (CPA) in a MCL xenograft model. We show that CPA administration before virus enhances oncolytic efficacy, likely through temporary immunosuppression. An interval of one-week between intravenous virus administration and fludarabine treatment further enhanced oncolysis, by synchronizing maximum prodrug convertase expression with fludarabine availability. Finally, three 23-day courses of triple sequential treatment with CPA, virus and fludarabine treatment resulted in complete regression of the xenografts. Secondary disease symptoms interfered with survival, but average survival times increased from 22 to 77 days. These studies document a reprogrammed oncolytic virus consolidating the effects of two chemotherapeutics, a concept well-suited for a phase I clinical trial for MCL patients for whom conventional therapies have failed. PMID:20686506

  17. Roles of cell signaling pathways in cell-to-cell contact-mediated Epstein-Barr virus transmission.

    PubMed

    Nanbo, Asuka; Terada, Haruna; Kachi, Kunihiro; Takada, Kenzo; Matsuda, Tadashi

    2012-09-01

    Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells. PMID:22718812

  18. Proteomic Analysis of Membrane Proteins of Vero Cells: Exploration of Potential Proteins Responsible for Virus Entry

    PubMed Central

    Guo, Donghua; Zhu, Qinghe; Zhang, Hong

    2014-01-01

    Vero cells are highly susceptible to many viruses in humans and animals, and its membrane proteins (MPs) are responsible for virus entry. In our study, the MP proteome of the Vero cells was investigated using a shotgun LC-MS/MS approach. Six hundred twenty-seven proteins, including a total of 1839 peptides, were identified in MP samples of the Vero cells. In 627 proteins, 307 proteins (48.96%) were annotated in terms of biological process of gene ontology (GO) categories; 356 proteins (56.78%) were annotated in terms of molecular function of GO categories; 414 proteins (66.03%) were annotated in terms of cellular components of GO categories. Of 627 identified proteins, seventeen proteins had been revealed to be virus receptor proteins. The resulting protein lists and highlighted proteins may provide valuable information to increase understanding of virus infection of Vero cells. PMID:24286161

  19. Green Fluorescent Protein-Tagged Retroviral Envelope Protein for Analysis of Virus-Cell Interactions

    PubMed Central

    Spitzer, Dirk; Dittmar, Kurt E. J.; Rohde, Manfred; Hauser, Hansjörg; Wirth, Dagmar

    2003-01-01

    Fluorescent retroviral envelope (Env) proteins were developed for direct visualization of viral particles. By fusing the enhanced green fluorescent protein (eGFP) to the N terminus of the amphotropic 4070A envelope protein, extracellular presentation of eGFP was achieved. Viruses incorporated the modified Env protein and efficiently infected cells. We used the GFP-tagged viruses for staining retrovirus receptor-positive cells, thereby circumventing indirect labeling techniques. By generating cells which conditionally expressed the GFP-tagged Env protein, we could confirm an inverse correlation between retroviral Env expression and infectivity (superinfection). eGFP-tagged virus particles are suitable for monitoring the dynamics of virus-cell interactions. PMID:12719600

  20. PARAMETERS DISTINGUISHING HERPES SIMPLEX VIRUS TYPE 2-TRANSFORMED TUMORIGENIC AND NONTUMORIGENIC RAT CELLS

    EPA Science Inventory

    A newly developed experimental model system was used to determine in vitro transformation-specific parameters which correlate with tumorigenicity. The data suggested that clonal herpes simplex virus type 2-transformed syngeneic rat embryo cells with intermediate, transformed rat ...

  1. Generation of a helper cell line for packaging avian leukosis virus-based vectors.

    PubMed Central

    Savatier, P; Bagnis, C; Thoraval, P; Poncet, D; Belakebi, M; Mallet, F; Legras, C; Cosset, F L; Thomas, J L; Chebloune, Y

    1989-01-01

    We constructed an avian leukosis virus-based packaging cell line, pHF-g, containing Rous-associated virus DNA with several alterations to abolish RNA packaging. One of them is a 52-base-pair deletion encompassing the putative encapsidation signal in the leader region. The 3' long terminal repeat was also removed and replaced by the polyadenylation sequence from the herpes simplex virus thymidine kinase gene. When pHF-g cells were transfected by an avian leukosis virus-based vector, they produced replication-defective virus at high titer but they did not release any replication-competent particles. Proviral DNA was shown to be correctly integrated as well as correctly expressed. Viral RNAs were shown to be correctly translated into gag-related polypeptides. Images PMID:2536089

  2. Noninvasive and label-free determination of virus infected cells by Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Moor, Kamila; Ohtani, Kiyoshi; Myrzakozha, Diyas; Zhanserkenova, Orik; Andriana, Bibin. B.; Sato, Hidetoshi

    2014-06-01

    The present study demonstrates that Raman spectroscopy is a powerful tool for the detection of virus-infected cells. Adenovirus infection of human embryonic kidney 293 cells was successfully detected at 12, 24, and 48 h after initiating the infection. The score plot of principal component analysis discriminated the spectra of the infected cells from those of the control cells. The viral infection was confirmed by the conventional immunostaining method performed 24 h after the infection. The newly developed method provides a fast and label-free means for the detection of virus-infected cells.

  3. Virus-Free Human Placental Cell Lines To Study Genetic Functions | NCI Technology Transfer Center | TTC

    Cancer.gov

    The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.The National Institute of Child Health and Human Development's Section on Cellular Differentiation is seeking statements of capability or interest from parties interested in collaborative research to further develop, evaluate, or commercialize immortalized virus-free human placental cell lines.

  4. Ebola virus disease in mice with transplanted human hematopoietic stem cells.

    PubMed

    Lüdtke, Anja; Oestereich, Lisa; Ruibal, Paula; Wurr, Stephanie; Pallasch, Elisa; Bockholt, Sabrina; Ip, Wing Hang; Rieger, Toni; Gómez-Medina, Sergio; Stocking, Carol; Rodríguez, Estefanía; Günther, Stephan; Muñoz-Fontela, César

    2015-04-01

    The development of treatments for Ebola virus disease (EVD) has been hampered by the lack of small-animal models that mimick human disease. Here we show that mice with transplanted human hematopoetic stem cells reproduce features typical of EVD. Infection with Ebola virus was associated with viremia, cell damage, liver steatosis, signs of hemorrhage, and high lethality. Our study provides a small-animal model with human components for the development of EVD therapies. PMID:25673711

  5. Single-cell genomics-based analysis of virus-host interactions in marine surface bacterioplankton.

    PubMed

    Labonté, Jessica M; Swan, Brandon K; Poulos, Bonnie; Luo, Haiwei; Koren, Sergey; Hallam, Steven J; Sullivan, Matthew B; Woyke, Tanja; Wommack, K Eric; Stepanauskas, Ramunas

    2015-11-01

    Viral infections dynamically alter the composition and metabolic potential of marine microbial communities and the evolutionary trajectories of host populations with resulting feedback on biogeochemical cycles. It is quite possible that all microbial populations in the ocean are impacted by viral infections. Our knowledge of virus-host relationships, however, has been limited to a minute fraction of cultivated host groups. Here, we utilized single-cell sequencing to obtain genomic blueprints of viruses inside or attached to individual bacterial and archaeal cells captured in their native environment, circumventing the need for host and virus cultivation. A combination of comparative genomics, metagenomic fragment recruitment, sequence anomalies and irregularities in sequence coverage depth and genome recovery were utilized to detect viruses and to decipher modes of virus-host interactions. Members of all three tailed phage families were identified in 20 out of 58 phylogenetically and geographically diverse single amplified genomes (SAGs) of marine bacteria and archaea. At least four phage-host interactions had the characteristics of late lytic infections, all of which were found in metabolically active cells. One virus had genetic potential for lysogeny. Our findings include first known viruses of Thaumarchaeota, Marinimicrobia, Verrucomicrobia and Gammaproteobacteria clusters SAR86 and SAR92. Viruses were also found in SAGs of Alphaproteobacteria and Bacteroidetes. A high fragment recruitment of viral metagenomic reads confirmed that most of the SAG-associated viruses are abundant in the ocean. Our study demonstrates that single-cell genomics, in conjunction with sequence-based computational tools, enable in situ, cultivation-independent insights into host-virus interactions in complex microbial communities. PMID:25848873

  6. Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

    PubMed

    Fujiwara, T.; Giesman-Cookmeyer, D.; Ding, B.; Lommel, S. A.; Lucas, W. J.

    1993-12-01

    Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread. PMID:12271056

  7. T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy

    PubMed Central

    Yu, Feng; Wang, Xingbing; Guo, Z Sheng; Bartlett, David L; Gottschalk, Stephen M; Song, Xiao-Tong

    2014-01-01

    Oncolytic vaccinia virus (VV) therapy has shown promise in preclinical models and in clinical studies. However, complete responses have rarely been observed. This lack of efficacy is most likely due to suboptimal virus spread through the tumor resulting in limited tumor cell destruction. We reasoned that redirecting T cells to the tumor has the potential to improve the antitumor activity of oncolytic VVs. We, therefore, constructed a VV encoding a secretory bispecific T-cell engager consisting of two single- chain variable fragments specific for CD3 and the tumor cell surface antigen EphA2 (EphA2-T-cell engager-armed VV (EphA2-TEA-VV)). In vitro, EphA2-TEA-VV's ability to replicate and induce oncolysis was similar to that of unmodified virus. However, only tumor cells infected with EphA2-TEA-VV induced T-cell activation as judged by the secretion of interferon-γ and interleukin-2. In coculture assays, EphA2-TEA-VV not only killed infected tumor cells, but in the presence of T cells, it also induced bystander killing of noninfected tumor cells. In vivo, EphA2-TEA-VV plus T cells had potent antitumor activity in comparison with control VV plus T cells in a lung cancer xenograft model. Thus, arming oncolytic VVs with T-cell engagers may represent a promising approach to improve oncolytic virus therapy. PMID:24135899

  8. Application of speckle dynamics for studying metabolic activity of cell cultures with herpes virus

    NASA Astrophysics Data System (ADS)

    Vladimirov, A. P.; Bakharev, A. A.; Malygin, A. S.; Mikhaylova, J. A.; Borodin, E. M.; Poryvayeva, A. P.; Glinskikh, N. P.

    2014-05-01

    The report considers the results of the experiments in which digital values of light intensity I and the image area correlation index η values were recorded on a real-time basis for one or two days. Three cell cultures with viruses along with intact cultures were investigated. High correlation of dependence of η values on time t values was demonstrated for three cultures. The η=η(t) and I=I(t) dependences for cells with and without viruses differ considerably. It was shown that the presence of viruses could be determined as early as ten minutes after measurements were started.

  9. Metabolic stress in infected cells may represent a therapeutic target for human immunodeficiency virus infection.

    PubMed

    Alonso-Villaverde, Carlos; Menéndez, Javier A; Joven, Jorge

    2013-07-01

    Worldwide, there are thousands of new cases of human immunodeficiency virus-1 (HIV-1) infection per day. The effectiveness of current combination antiretroviral therapy (ART) is relative; to prioritize finding vaccines and/or cure-oriented initiatives should be reinforced because there is little room, if any, for procrastination. Basic and clinical findings on HIV-1 reservoirs suggest that disruption of virus latency is feasible. Because the goal is curing HIV-1 infection, we should be aware that the challenge is to eradicate the viruses of every single infected cell and consequently acting upon virus latency is necessary but not sufficient. The large majority of the virus reservoir, CD4(+) T lymphocytes, is readily accessible but other minor reservoirs, where ART does not diffuse, require innovative strategies. The situation closely resembles that currently faced in the treatment of cancer. Exploiting the fact that histone deacetylase inhibitors, mainly vorinostat, may disrupt the latency of HIV-1, we propose to supplement this effect with a programmed interference in the metabolic stress of infected cells. Metformin and chloroquine are cheap and accessible modulators of pro-survival mechanisms to which viruses are constantly confronted to generate alternative energy sources and maximize virus production. Metformin restrains the use of the usurped cellular biosynthetic machinery by viral genes and chloroquine contributes to death of infected cells. We suggest that the combination of vorinostat, chloroquine and metformin should be combined with ART to pursue viral eradication in infected cells. PMID:23639282

  10. Expression of Epstein-Barr virus genes in different cell types after microinjection of viral DNA.

    PubMed Central

    Graessmann, A; Wolf, H; Bornkamm, G W

    1980-01-01

    Gene expression of Epstein-Barr virus (EBV) was studied after microinjection of viral DNA into different types of cells. Raji TK- cells, known to express viral gene functions after superinfection with the EBV-P3HR-1 virus strain, were attached to plastic dishes by using anti-lymphocyte IgG, phytohemagglutinin, or concanavalin A as a ligand. It was difficult to inject DNA into the small and fragile Raji cells. After formation of polykaryons by cell fusion, microinjection became more efficient. At 24 hr after injection of P3HR-1 virus DNA, 90-100% of the injected cells expressed the early antigen complex as observed by immunofluorescence staining; 70-80% of the cells simultaneously incorported [3H]thymidine, indicating that thymidine kinase is expressed after injection of viral DNA. Additionally, synthesis of the virus capsid antigen was demonstrated in 20-30% of the recipient Raji cells. Human diploid fibroblasts, African green monkey kidney cells, and rat fibroblasts, which do not represent natural target cells for EBV, could also be induced to synthesis of early antigen complex by injection of P3HR-1 virus DNA. Images PMID:6244558

  11. CD9, a tetraspan transmembrane protein, renders cells susceptible to canine distemper virus.

    PubMed Central

    Löffler, S; Lottspeich, F; Lanza, F; Azorsa, D O; ter Meulen, V; Schneider-Schaulies, J

    1997-01-01

    Canine distemper virus (CDV), a lymphotropic and neurotropic negative-stranded RNA virus of the Morbillivirus genus, causes a life-threatening disease in several carnivores, including domestic dogs. To identify the cellular receptor(s) involved in the uptake of CDV by susceptible cells, we isolated a monoclonal antibody (MAb K41) which binds to the cell surface and inhibits the CDV infection of several cell lines from various species. Pretreatment of cells with MAb K41 reduces the number of infectious centers and the size of the syncytia. Using affinity chromatography with MAb K41, we purified from HeLa and Vero cell extracts a 26-kDa protein which contained the amino acid sequence TKDEPQRETLK of human CD9, a member of the tetraspan transmembrane or transmembrane 4 superfamily of cell surface proteins. Transfection of NIH 3T3 or MDBK cells with a CD9 expression plasmid rendered these cells permissive for viral infection and raised virus production by a factor of 10 to 100. The mechanism involved is still unclear, since we were unable to detect direct binding of CDV to CD9 by using immunoprecipitation and a virus overlay protein binding assay. These findings indicate that human CD9 and its homologs in other species are necessary factors for the uptake of CDV by target cells, the formation of syncytia, and the production of progeny virus. PMID:8985321

  12. Chimeric antigen receptor–engineered T cells as oncolytic virus carriers

    PubMed Central

    VanSeggelen, Heather; Tantalo, Daniela GM; Afsahi, Arya; Hammill, Joanne A; Bramson, Jonathan L

    2015-01-01

    The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments. PMID:27119109

  13. The role of cell-associated virus in mother-to-child HIV transmission.

    PubMed

    Milligan, Caitlin; Overbaugh, Julie

    2014-12-15

    Mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) continues to contribute to the global burden of disease despite great advances in antiretroviral (ARV) treatment and prophylaxis. In this review, we discuss the proposed mechanisms of MTCT, evidence for cell-free and cell-associated transmission in different routes of MTCT, and the impact of ARVs on virus levels and transmission. Many population-based studies support a role for cell-associated virus in transmission and in vitro studies also provide some support for this mode of transmission. However, animal model studies provide proof-of-principle that cell-free virus can establish infection in infants, and studies of ARVs in HIV-infected pregnant women show a strong correlation with reduction in cell-free virus levels and protection. ARV treatment in MTCT potentially provides opportunities to better define the infectious form of virus, but these studies will require better tools to measure the infectious cell reservoir. PMID:25414417

  14. Re-examination of the relationship between marine virus and microbial cell abundances.

    PubMed

    Wigington, Charles H; Sonderegger, Derek; Brussaard, Corina P D; Buchan, Alison; Finke, Jan F; Fuhrman, Jed A; Lennon, Jay T; Middelboe, Mathias; Suttle, Curtis A; Stock, Charles; Wilson, William H; Wommack, K Eric; Wilhelm, Steven W; Weitz, Joshua S

    2016-01-01

    Marine viruses are critical drivers of ocean biogeochemistry, and their abundances vary spatiotemporally in the global oceans, with upper estimates exceeding 10(8) per ml. Over many years, a consensus has emerged that virus abundances are typically tenfold higher than microbial cell abundances. However, the true explanatory power of a linear relationship and its robustness across diverse ocean environments is unclear. Here, we compile 5,671 microbial cell and virus abundance estimates from 25 distinct marine surveys and find substantial variation in the virus-to-microbial cell ratio, in which a 10:1 model has either limited or no explanatory power. Instead, virus abundances are better described as nonlinear, power-law functions of microbial cell abundances. The fitted scaling exponents are typically less than 1, implying that the virus-to-microbial cell ratio decreases with microbial cell density, rather than remaining fixed. The observed scaling also implies that viral effect sizes derived from 'representative' abundances require substantial refinement to be extrapolated to regional or global scales. PMID:27572161

  15. EXPRESSION OF THE MAIZE MOSAIC VIRUS GLYCOPROTEIN IN INSECT CELLS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize mosaic virus (genus Nucleorhabdovirus, family Rhabdoviridae) is transmitted in a persistent-propagative manner by Peregrinus maidis, the corn planthopper. Like other rhabdoviruses, the MMV genome encodes a surface glycoprotein that is likely involved in virus attachment and entry into host ce...

  16. GAPDH-A Recruits a Plant Virus Movement Protein to Cortical Virus Replication Complexes to Facilitate Viral Cell-to-Cell Movement

    PubMed Central

    Kaido, Masanori; Abe, Kazutomo; Mine, Akira; Hyodo, Kiwamu; Taniguchi, Takako; Taniguchi, Hisaaki; Mise, Kazuyuki; Okuno, Tetsuro

    2014-01-01

    The formation of virus movement protein (MP)-containing punctate structures on the cortical endoplasmic reticulum is required for efficient intercellular movement of Red clover necrotic mosaic virus (RCNMV), a bipartite positive-strand RNA plant virus. We found that these cortical punctate structures constitute a viral replication complex (VRC) in addition to the previously reported aggregate structures that formed adjacent to the nucleus. We identified host proteins that interacted with RCNMV MP in virus-infected Nicotiana benthamiana leaves using a tandem affinity purification method followed by mass spectrometry. One of these host proteins was glyceraldehyde 3-phosphate dehydrogenase-A (NbGAPDH-A), which is a component of the Calvin-Benson cycle in chloroplasts. Virus-induced gene silencing of NbGAPDH-A reduced RCNMV multiplication in the inoculated leaves, but not in the single cells, thereby suggesting that GAPDH-A plays a positive role in cell-to-cell movement of RCNMV. The fusion protein of NbGAPDH-A and green fluorescent protein localized exclusively to the chloroplasts. In the presence of RCNMV RNA1, however, the protein localized to the cortical VRC as well as the chloroplasts. Bimolecular fluorescence complementation assay and GST pulldown assay confirmed in vivo and in vitro interactions, respectively, between the MP and NbGAPDH-A. Furthermore, gene silencing of NbGAPDH-A inhibited MP localization to the cortical VRC. We discuss the possible roles of NbGAPDH-A in the RCNMV movement process. PMID:25411849

  17. Human T-cell lymphotropic virus in volunteer blood donors.

    PubMed

    Taylor, P E; Stevens, C E; Pindyck, J; Schrode, J; Steaffens, J W; Lee, H

    1990-01-01

    Serum samples collected in 1985 and 1986 from 18,257 donors to the Greater New York Blood Program were screened by enzyme-linked immunoassay for antibody to human T-cell lymphotropic virus (anti-HTLV). Fifteen samples (0.08%) were confirmed positive: 7 by radioimmunoprecipitation assay (RIPA) alone, 6 by Western blot alone, and 2 by combined results from both tests. One donor, whose original test result was uninterpretable because multiple nonspecific bands were present on RIPA, clearly tested positive on subsequent specimens. Follow-up testing of individuals with this type of result may be needed to resolve their HTLV status. Anti-HTLV prevalence increased with age and was significantly more common in black or Hispanic donors and in those born in the Caribbean than in other donors. All anti-HTLV-positive donors were negative for antibody to HIV-1, and only one donor (7% of those positive) would have been excluded by any of the routine donor screening tests used at that time. PMID:2173176

  18. Black Beetle Virus: Propagation in Drosophila Line 1 Cells and an Infection-Resistant Subline Carrying Endogenous Black Beetle Virus-Related Particles

    PubMed Central

    Friesen, Paul; Scotti, Paul; Longworth, John; Rueckert, Roland

    1980-01-01

    Black beetle virus (BBV), one of a recently discovered class of viruses with a bipartite genome, multiplied readily in Schneider's line 1 of Drosophila cells. Virus yields, on the order of 100 mg per liter of culture, were unusually high and represented some 20% of the total cell protein within 3 days after infection. A derivative subline of these Drosophila cells was found to be resistant to infection by BBV. These resistant cells were also found to carry small amounts of BBV-related particles, possibly a maturation-defective form of BBV. PMID:16789201

  19. Synthesis of herpes simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphonoacetic acid.

    PubMed Central

    Bolden, A; Aucker, J; Weissbach, A

    1975-01-01

    Purified nuclei, isolated from appropriately infected HeLa cells, are shown to synthesize large amounts of either herpes simplex virus (HSV) or vaccinia virus DNA in vitro. The rate of synthesis of DNA by nuclei from infected cells is up to 30 times higher than the synthesis of host DNA in vitro by nuclei isolated from uninfected HeLa cells. Thus HSV nuclei obtained from HSV-infected cells make DNA in vitro at a rate comparable to that seen in the intact, infected cell. Molecular hybridization studies showed that 80% of the DNA sequences synthesized in vitro by nuclei from herpesvirus-infected cells are herpesvirus specific. Vaccinia virus nuclei from vaccinia virus-infected cells, also produce comparable percentages of vaccinia virus-specific DNA sequences. Adenovirus nuclei from adenovirus 2-infected HeLa cells, which also synthesize viral DNA in vitro, have been included in this study. Synthesis of DNA by HSV or vaccinia virus nuclei is markedly inhibited by the corresponding viral-specific antisera. These antisera inhibit in a similar fashion the purified herpesvirus-induced or vaccinia virus-induced DNA polymerase isolated from infected cells. Phosphonoacetic acid, reported to be a specific inhibitor of herpesvirus formation and the herpesvirus-induced DNA polymerase, is equally effective as an inhibitor of HSV DNA synthesis in isolated nuclei in vitro. However, we also find phosphonoacetic acid to be an effective inhibitor of vaccinia virus nuclear DNA synthesis and the purified vaccinia virus-induced DNA polymerase. In addition, this compound shows significant inhibition of DNA synthesis in isolated nuclei obtained from adenovirus-infected or uninfected cells and is a potent inhibitor of HeLa cell DNA polymerase alpha. PMID:172658

  20. Modes of Human T Cell Leukemia Virus Type 1 Transmission, Replication and Persistence

    PubMed Central

    Carpentier, Alexandre; Barez, Pierre-Yves; Hamaidia, Malik; Gazon, Hélène; de Brogniez, Alix; Perike, Srikanth; Gillet, Nicolas; Willems, Luc

    2015-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus that causes cancer (Adult T cell Leukemia, ATL) and a spectrum of inflammatory diseases (mainly HTLV-associated myelopathy—tropical spastic paraparesis, HAM/TSP). Since virions are particularly unstable, HTLV-1 transmission primarily occurs by transfer of a cell carrying an integrated provirus. After transcription, the viral genomic RNA undergoes reverse transcription and integration into the chromosomal DNA of a cell from the newly infected host. The virus then replicates by either one of two modes: (i) an infectious cycle by virus budding and infection of new targets and (ii) mitotic division of cells harboring an integrated provirus. HTLV-1 replication initiates a series of mechanisms in the host including antiviral immunity and checkpoint control of cell proliferation. HTLV-1 has elaborated strategies to counteract these defense mechanisms allowing continuous persistence in humans. PMID:26198240

  1. Natural killer cell dysfunction during acute infection with foot-and-mouth diseaase virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural killer cells (NK) provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. The role of these cells in foot-and-mouth disease virus (FMDV) infection is unknown. Previously, we characterized the phenotype and function of NK cells fr...

  2. Infection of polarized MDCK cells with herpes simplex virus 1: two asymmetrically distributed cell receptors interact with different viral proteins.

    PubMed Central

    Sears, A E; McGwire, B S; Roizman, B

    1991-01-01

    Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable. Images PMID:1647025

  3. Infection of Polarized MDCK Cells with Herpes Simplex Virus 1: Two Asymmetrically Distributed Cell Receptors Interact with Different Viral Proteins

    NASA Astrophysics Data System (ADS)

    Sears, Amy E.; McGwire, Bradford S.; Roizman, Bernard

    1991-06-01

    Herpes simplex virus 1 attaches to at least two cell surface receptors. In polarized epithelial (Madin-Darby canine kidney; MDCK) cells one receptor is located in the apical surface and attachment to the cells requires the presence of glycoprotein C in the virus. The second receptor is located in the basal surface and does not require the presence of glycoprotein C. Exposure of MDCK cells at either the apical or basal surface to wild-type virus yields plaques and viral products whereas infection by a glycoprotein C-negative mutant yields identical results only after exposure of MDCK cells to virus at the basal surface. Multiple receptors for viral entry into cells expand the host range of the virus. The observation that glycoprotein C-negative mutants are infectious in many nonpolarized cell lines suggests that cells in culture may express more than one receptor and explains why genes that specify the viral proteins that recognize redundant receptors, like glycoprotein C, are expendable.

  4. Replication and supercoiling of simian virus 40 DNA in cell extracts from human cells.

    PubMed Central

    Stillman, B W; Gluzman, Y

    1985-01-01

    Soluble extracts prepared from the nucleus and cytoplasm of human 293 cells are capable of efficient replication and supercoiling of added DNA templates that contain the origin of simian virus 40 replication. Extracts prepared from human HeLa cells are less active than similarly prepared extracts from 293 cells for initiation and elongation of nascent DNA strands. DNA synthesis is dependent on addition of purified simian virus 40 tumor (T) antigen, which is isolated by immunoaffinity chromatography of extracts from cells infected with an adenovirus modified to produce large quantities of this protein. In the presence of T antigen and the cytoplasmic extract, replication initiates at the origin and continues bidirectionally. Initiation is completely dependent on functional origin sequences; a plasmid DNA containing an origin mutation known to affect DNA replication in vivo fails to replicate in vitro. Multiple rounds of DNA synthesis occur, as shown by the appearance of heavy-heavy, bromodeoxyuridine-labeled DNA products. The products of this reaction are resolved, but are relaxed, covalently closed DNA circles. Addition of a nuclear extract during DNA synthesis promotes the negative supercoiling of the replicated DNA molecules. Images PMID:3018548

  5. Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

    PubMed Central

    Langhoff, E; Terwilliger, E F; Bos, H J; Kalland, K H; Poznansky, M C; Bacon, O M; Haseltine, W A

    1991-01-01

    The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection. Images PMID:1910172

  6. Characterization of the env gene and long terminal repeat of molecularly cloned Friend mink cell focus-inducing virus DNA.

    PubMed Central

    Adachi, A; Sakai, K; Kitamura, N; Nakanishi, S; Niwa, O; Matsuyama, M; Ishimoto, A

    1984-01-01

    The highly oncogenic erythroleukemia-inducing Friend mink cell focus-inducing (MCF) virus was molecularly cloned in phage lambda gtWES.lambda B, and the DNA sequences of the env gene and the long terminal repeat were determined. The nucleotide sequences of Friend MCF virus and Friend spleen focus-forming virus were quite homologous, supporting the hypothesis that Friend spleen focus-forming virus might be generated via Friend MCF virus from an ecotropic Friend virus mainly by some deletions. Despite their different pathogenicity, the nucleotide sequences of the env gene of Friend MCF virus and Moloney MCF virus were quite homologous, suggesting that the putative parent sequence for the generation of both MCF viruses and the recombinational mechanism for their generation might be the same. We compare the amino acid sequences in lymphoid leukemia-inducing ecotropic Moloney virus and Moloney MCF virus, and erythroblastic leukemia-inducing ecotropic Friend virus, Friend-MCF virus, and Friend spleen focus-forming virus. The Friend MCF virus long terminal repeat was found to be 550 base pairs long. This contained two copies of the 39-base-pair tandem repeat, whereas the spleen focus-forming virus genome contained a single copy of the same sequence. PMID:6328011

  7. Bumblebee size polymorphism and worker response to queen pheromone.

    PubMed

    Holman, Luke

    2014-01-01

    Queen pheromones are chemical signals produced by reproductive individuals in social insect colonies. In many species they are key to the maintenance of reproductive division of labor, with workers beginning to reproduce individually once the queen pheromone disappears. Recently, a queen pheromone that negatively affects worker fecundity was discovered in the bumblebee Bombus terrestris, presenting an exciting opportunity for comparisons with analogous queen pheromones in independently-evolved eusocial lineages such as honey bees, ants, wasps and termites. I set out to replicate this discovery and verify its reproducibility. Using blind, controlled experiments, I found that n-pentacosane (C25) does indeed negatively affect worker ovary development. Moreover, the pheromone affects both large and small workers, and applies to workers from large, mature colonies as well as young colonies. Given that C25 is readily available and that bumblebees are popular study organisms, I hope that this replication will encourage other researchers to tackle the many research questions enabled by the discovery of a queen pheromone. PMID:25289189

  8. Do sexist organizational cultures create the Queen Bee?

    PubMed

    Derks, Belle; Ellemers, Naomi; van Laar, Colette; de Groot, Kim

    2011-09-01

    'Queen Bees' are senior women in masculine organizational cultures who have fulfilled their career aspirations by dissociating themselves from their gender while simultaneously contributing to the gender stereotyping of other women. It is often assumed that this phenomenon contributes to gender discrimination in organizations, and is inherent to the personalities of successful career women. We argue for a social identity explanation and examine organizational conditions that foster the Queen Bee phenomenon. Participants were 94 women holding senior positions in diverse companies in The Netherlands who participated in an on-line survey. In line with predictions, indicators of the Queen Bee phenomenon (increased gender stereotyping and masculine self-descriptions) were found mostly among women who indicated they had started their career with low gender identification and who had subsequently experienced a high degree of gender discrimination on their way up. By contrast, the experience of gender discrimination was unrelated to signs of the Queen Bee phenomenon among women who indicated to be highly identified when they started their career. Results are discussed in light of social identity theory, interpreting the Queen Bee phenomenon as an individual mobility response of low gender identified women to the gender discrimination they encounter in their work. PMID:21884548

  9. The evolution of queen pheromones in the ant genus Lasius.

    PubMed

    Holman, L; Lanfear, R; d'Ettorre, P

    2013-07-01

    Queen pheromones are among the most important chemical messages regulating insect societies yet they remain largely undiscovered, hindering research into interesting proximate and ultimate questions. Identifying queen pheromones in multiple species would give new insight into the selective pressures and evolutionary constraints acting on these ubiquitous signals. Here, we present experimental and comparative evidence that 3-methylalkanes, hydrocarbons present on the queen's cuticle, are a queen pheromone throughout the ant genus Lasius. Interspecific variation in the chemical profile is consistent with 3-methylalkanes evolving more slowly than other types of hydrocarbons, perhaps due to differential selection or evolutionary constraints. We argue that the sensory ecology of the worker response imposes strong stabilizing selection on queen pheromones relative to other hydrocarbons. 3-Methylalkanes are also strongly physiologically and genetically coupled with fecundity in at least one Lasius species, which may translate into evolutionary constraints. Our results highlight how honest signalling could minimize evolutionary conflict over reproduction, promoting the evolution and maintenance of eusociality. PMID:23662630

  10. Bumblebee size polymorphism and worker response to queen pheromone

    PubMed Central

    2014-01-01

    Queen pheromones are chemical signals produced by reproductive individuals in social insect colonies. In many species they are key to the maintenance of reproductive division of labor, with workers beginning to reproduce individually once the queen pheromone disappears. Recently, a queen pheromone that negatively affects worker fecundity was discovered in the bumblebee Bombus terrestris, presenting an exciting opportunity for comparisons with analogous queen pheromones in independently-evolved eusocial lineages such as honey bees, ants, wasps and termites. I set out to replicate this discovery and verify its reproducibility. Using blind, controlled experiments, I found that n-pentacosane (C25) does indeed negatively affect worker ovary development. Moreover, the pheromone affects both large and small workers, and applies to workers from large, mature colonies as well as young colonies. Given that C25 is readily available and that bumblebees are popular study organisms, I hope that this replication will encourage other researchers to tackle the many research questions enabled by the discovery of a queen pheromone. PMID:25289189

  11. Presence of Nosema ceranae associated with honeybee queen introductions.

    PubMed

    Muñoz, Irene; Cepero, Almudena; Pinto, Maria Alice; Martín-Hernández, Raquel; Higes, Mariano; De la Rúa, Pilar

    2014-04-01

    Microsporidiosis caused by Nosema species is one of the factors threatening the health of the honeybee (Apis mellifera), which is an essential element in agriculture mainly due to its pollination function. The dispersion of this pathogen may be influenced by many factors, including various aspects of beekeeping management such as introduction of queens with different origin. Herein we study the relation of the presence and distribution of Nosema spp. and the replacement of queens in honeybee populations settled on the Atlantic Canary Islands. While Nosema apis has not been detected, an increase of the presence and distribution of Nosema ceranae during the last decade has been observed in parallel with a higher frequency of foreign queens. On the other hand, a reduction of the number of N. ceranae positive colonies was observed on those islands with continued replacement of queens. We suggest that such replacement could help maintaining low rates of Nosema infection, but healthy queens native to these islands should be used in order to conserve local honeybee diversity. PMID:24568841

  12. The Red Queen lives: Epistasis between linked resistance loci.

    PubMed

    Metzger, César M J A; Luijckx, Pepijn; Bento, Gilberto; Mariadassou, Mahendra; Ebert, Dieter

    2016-02-01

    A popular theory explaining the maintenance of genetic recombination (sex) is the Red Queen Theory. This theory revolves around the idea that time-lagged negative frequency-dependent selection by parasites favors rare host genotypes generated through recombination. Although the Red Queen has been studied for decades, one of its key assumptions has remained unsupported. The signature host-parasite specificity underlying the Red Queen, where infection depends on a match between host and parasite genotypes, relies on epistasis between linked resistance loci for which no empirical evidence exists. We performed 13 genetic crosses and tested over 7000 Daphnia magna genotypes for resistance to two strains of the bacterial pathogen Pasteuria ramosa. Results reveal the presence of strong epistasis between three closely linked resistance loci. One locus masks the expression of the other two, while these two interact to produce a single resistance phenotype. Changing a single allele on one of these interacting loci can reverse resistance against the tested parasites. Such a genetic mechanism is consistent with host and parasite specificity assumed by the Red Queen Theory. These results thus provide evidence for a fundamental assumption of this theory and provide a genetic basis for understanding the Red Queen dynamics in the Daphnia-Pasteuria system. PMID:26763092

  13. Shared catalysis in virus entry and bacterial cell wall depolymerization.

    PubMed

    Cohen, Daniel N; Sham, Yuk Y; Haugstad, Greg D; Xiang, Ye; Rossmann, Michael G; Anderson, Dwight L; Popham, David L

    2009-04-01

    Bacterial virus entry and cell wall depolymerization require the breakdown of peptidoglycan (PG), the peptide-cross-linked polysaccharide matrix that surrounds bacterial cells. Structural studies of lysostaphin, a PG lytic enzyme (autolysin), have suggested that residues in the active site facilitate hydrolysis, but a clear mechanism for this reaction has remained unsolved. The active-site residues and a structural pattern of beta-sheets are conserved among lysostaphin homologs (such as LytM of Staphylococcus aureus) and the C-terminal domain of gene product 13 (gp13), a protein at the tail tip of the Bacillus subtilis bacteriophage varphi29. gp13 activity on PG and muropeptides was assayed using high-performance liquid chromatography, and gp13 was found to be a d,d-endopeptidase that cleaved the peptide cross-link. Computational modeling of the B. subtilis cross-linked peptide into the gp13 active site suggested that Asp195 may facilitate scissile-bond activation and that His247 is oriented to mediate nucleophile generation. To our knowledge, this is the first model of a Zn(2)(+) metallopeptidase and its substrate. Residue Asp195 of gp13 was found to be critical for Zn(2)(+) binding and catalysis by substitution mutagenesis with Ala or Cys. Circular dichroism and particle-induced X-ray emission spectroscopy showed that the general protein folding and Zn(2)(+) binding were maintained in the Cys mutant but reduced in the Ala mutant. These findings together support a model in which the Asp195 and His247 in gp13 and homologous residues in the LytM and lysostaphin active sites facilitate hydrolysis of the peptide substrate that cross-links PG. Thus, these autolysins and phage-entry enzymes have a shared chemical mechanism of action. PMID:19361422

  14. Venom alkaloid and cuticular hydrocarbon profiles are associated with social organization, queen fertility status, and queen genotype in the fire ant Solenopsis invicta.

    PubMed

    Eliyahu, Dorit; Ross, Kenneth G; Haight, Kevin L; Keller, Laurent; Liebig, Jürgen

    2011-11-01

    Queens in social insect colonies advertise their presence in the colony to: a) attract workers' attention and care; b) gain acceptance by workers as replacement or supplemental reproductives; c) prevent reproductive development in nestmates. We analyzed the chemical content of whole body surface extracts of adult queens of different developmental and reproductive stages, and of adult workers from monogyne (single colony queen) and polygyne (multiple colony queens) forms of the fire ant Solenopsis invicta. We found that the composition of the most abundant components, venom alkaloids, differed between queens and workers, as well as between reproductive and non-reproductive queens. Additionally, workers of the two forms could be distinguished by alkaloid composition. Finally, sexually mature, non-reproductive queens from polygyne colonies differed in their proportions of cis-piperidine alkaloids, depending on their Gp-9 genotype, although the difference disappeared once they became functional reproductives. Among the unsaturated cuticular hydrocarbons characteristic of queens, there were differences in amounts of alkenes/alkadienes between non-reproductive polygyne queens of different Gp-9 genotypes, between non-reproductive and reproductive queens, and between polygyne and monogyne reproductive queens, with the amounts increasing at a relatively higher rate through reproductive ontogeny in queens bearing the Gp-9 b allele. Given that the genotype-specific piperidine differences reflect differences in rates of reproductive maturation between queens, we speculate that these abundant and unique compounds have been co-opted to serve in fertility signaling, while the cuticular hydrocarbons now play a complementary role in regulation of social organization by signaling queen Gp-9 genotype. PMID:22095515

  15. The value of oviposition timing, queen presence and kinship in a social insect

    PubMed Central

    Ozan, Martina; Helanterä, Heikki; Sundström, Liselotte

    2013-01-01

    Reproductive cooperation confers benefits, but simultaneously creates conflicts among cooperators. Queens in multi-queen colonies of ants share a nest and its resources, but reproductive competition among queens often results in unequal reproduction. Two mutually non-exclusive factors may produce such inequality in reproduction: worker intervention or queen traits. Workers may intervene by favouring some queens over others, owing to either kinship or queen signals. Queens may differ in their intrinsic fecundity at the onset of oviposition or in their timing of the onset of oviposition, leading to their unequal representation in the brood. Here, we test the role of queen kin value (relatedness) to workers, timing of the onset of oviposition and signals of presence by queens in determining the maternity of offspring. We show that queens of the ant Formica fusca gained a significantly higher proportion of sexuals in the brood when ovipositing early, and that the presence of a caged queen resulted in a significant increase in both her share of sexual brood and her overall reproductive share. Moreover, the lower the kin value of the queen, the more the workers invested in their own reproduction by producing males. Our results show that both kinship and breeding phenology influence the outcome of reproductive conflicts, and the balance of direct and indirect fitness benefits in the multi-queen colonies of F. fusca. PMID:23843391

  16. The role of queens in colonies of the swarm-founding wasp Parachartergus colobopterus.

    PubMed

    Herman; Queller; Strassmann

    2000-04-01

    Social insect queens reproduce while workers generally do not. Queens may also have other behavioural roles in the colony. In small, independent-founding colonies of social wasps, the dominant queen physically enforces her interests over those of the workers and serves as a pacemaker of the colony, stimulating workers to forage and engage in other tasks. By contrast, in large-colony, swarm-founding wasps, the collective interests of the workers are fulfilled in sex allocation and production of males, whether or not they coincide with the interests of the queens. The behavioural role of the queens in such species has not been extensively studied. We investigated the role of the queens both in regulating worker activity and in reducing the numbers of reproductively active queens in the swarm-founding epiponine wasp Parachartergus colobopterus. We found no evidence that queens regulate worker activity, as they were rarely involved in any interactions. Worker activity may be self-organized, without centralized active control by anyone. Furthermore, we found no evidence that the reduction in queen number characteristic of this tribe of wasps occurs in response to aggression among queens. The reduction in queen number may be a result of worker treatment of queens, although worker discrimination against some queens was not obvious in our data. i Copyright 2000 The Association for the Study of Animal Behaviour. PMID:10792939

  17. How antibodies alter the cell entry pathway of dengue virus particles in macrophages

    PubMed Central

    Ayala-Nunez, Nilda V.; Hoornweg, Tabitha E.; van de Pol, Denise P.I.; Sjollema, Klaas A.; Flipse, Jacky; van der Schaar, Hilde M.; Smit, Jolanda M.

    2016-01-01

    Antibody-dependent enhancement of dengue virus (DENV) infection plays an important role in the exacerbation of DENV-induced disease. To understand how antibodies influence the fate of DENV particles, we explored the cell entry pathway of DENV in the absence and presence of antibodies in macrophage-like P388D1 cells. Recent studies unraveled that both mature and immature DENV particles contribute to ADE, hence, both particles were studied. We observed that antibody-opsonized DENV enters P388D1 cells through a different pathway than non-opsonized DENV. Antibody-mediated DENV entry was dependent on FcγRs, pH, Eps15, dynamin, actin, PI3K, Rab5, and Rab7. In the absence of antibodies, DENV cell entry was FcγR, PI3K, and Rab5-independent. Live-cell imaging of fluorescently-labeled particles revealed that actin-mediated membrane protrusions facilitate virus uptake. In fact, actin protrusions were found to actively search and capture antibody-bound virus particles distantly located from the cell body, a phenomenon that is not observed in the absence of antibodies. Overall, similar results were seen for antibody-opsonized standard and antibody-bound immature DENV preparations, indicating that the maturation status of the virus does not control the entry pathway. Collectively, our findings suggest that antibodies alter the cell entry pathway of DENV and trigger a novel mechanism of initial virus-cell contact. PMID:27385443

  18. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance.

    PubMed

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J; Butler, Mark S; Edwards, David J; Fry, Scott R; Bermingham, Imogen M; Cooper, Matthew A; Young, Paul R

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  19. A generic screening platform for inhibitors of virus induced cell fusion using cellular electrical impedance

    PubMed Central

    Watterson, Daniel; Robinson, Jodie; Chappell, Keith J.; Butler, Mark S.; Edwards, David J.; Fry, Scott R.; Bermingham, Imogen M.; Cooper, Matthew A.; Young, Paul R.

    2016-01-01

    Fusion of the viral envelope with host cell membranes is an essential step in the life cycle of all enveloped viruses. Despite such a clear target for antiviral drug development, few anti-fusion drugs have progressed to market. One significant hurdle is the absence of a generic, high-throughput, reproducible fusion assay. Here we report that real time, label-free measurement of cellular electrical impedance can quantify cell-cell fusion mediated by either individually expressed recombinant viral fusion proteins, or native virus infection. We validated this approach for all three classes of viral fusion and demonstrated utility in quantifying fusion inhibition using antibodies and small molecule inhibitors specific for dengue virus and respiratory syncytial virus. PMID:26976324

  20. Cell-Free Transmission and In Vivo Replication of Marek's Disease Virus 1

    PubMed Central

    Nazerian, K.; Witter, R. L.

    1970-01-01

    Marek's disease virus recovered from the feather follicle of infected chickens was found to be infectious for chickens in cell-free preparations. The virus replicated in epithelial cells of the germinative layer of the feather follicle epidermis, producing both intranuclear and round or diffuse cytoplasmic inclusion bodies in the infected cells. It was found at this site 2 weeks postinoculation and prior to the development of tumor or other gross lesions. In the nucleus, many naked and a few enveloped herpesvirions were found, whereas the cytoplasm contained predominantly enveloped herpesvirions, which were usually within the cytoplasmic inclusion bodies. Approximately 80% of the extracellular virions were enveloped. Studies with both virulent and avirulent strains of the virus revealed a relationship between virulence, contagiousness, and replication of the virus in the feather follicle. Images PMID:4191324

  1. Mass spectrometric analysis of host cell proteins interacting with dengue virus nonstructural protein 1 in dengue virus-infected HepG2 cells.

    PubMed

    Dechtawewat, Thanyaporn; Paemanee, Atchara; Roytrakul, Sittiruk; Songprakhon, Pucharee; Limjindaporn, Thawornchai; Yenchitsomanus, Pa-Thai; Saitornuang, Sawanan; Puttikhunt, Chunya; Kasinrerk, Watchara; Malasit, Prida; Noisakran, Sansanee

    2016-09-01

    Dengue virus (DENV) infection is a leading cause of the mosquito-borne infectious diseases that affect humans worldwide. Virus-host interactions appear to play significant roles in DENV replication and the pathogenesis of DENV infection. Nonstructural protein 1 (NS1) of DENV is likely involved in these processes; however, its associations with host cell proteins in DENV infection remain unclear. In this study, we used a combination of techniques (immunoprecipitation, in-solution trypsin digestion, and LC-MS/MS) to identify the host cell proteins that interact with cell-associated NS1 in an in vitro model of DENV infection in the human hepatocyte HepG2 cell line. Thirty-six novel host cell proteins were identified as potential DENV NS1-interacting partners. A large number of these proteins had characteristic binding or catalytic activities, and were involved in cellular metabolism. Coimmunoprecipitation and colocalization assays confirmed the interactions of DENV NS1 and human NIMA-related kinase 2 (NEK2), thousand and one amino acid protein kinase 1 (TAO1), and component of oligomeric Golgi complex 1 (COG1) proteins in virus-infected cells. This study reports a novel set of DENV NS1-interacting host cell proteins in the HepG2 cell line and proposes possible roles for human NEK2, TAO1, and COG1 in DENV infection. PMID:27108190

  2. Active and Selective Transcytosis of Cell-Free Human Immunodeficiency Virus through a Tight Polarized Monolayer of Human Endometrial Cells

    PubMed Central

    Hocini, Hakim; Becquart, Pierre; Bouhlal, Hicham; Chomont, Nicolas; Ancuta, Petronela; Kazatchkine, Michel D.; Bélec, Laurent

    2001-01-01

    We report that both primary and laboratory-adapted infectious human immunodeficiency virus type 1 (HIV-1) isolates in a cell-free form are capable of transcytosis through a tight and polarized monolayer of human endometrial cells. Trancytosis of cell-free HIV occurs in a strain-selective fashion and appears to be dependent on interactions between HIV envelope glycoproteins and lectins on the apical membrane of the epithelial cells. These findings provide new insights into the initial events occurring during heterosexual transmission of the virus. PMID:11333919

  3. Movement of rice yellow mottle virus between xylem cells through pit membranes

    PubMed Central

    Opalka, Natacha; Brugidou, Christophe; Bonneau, Caroline; Nicole, Michel; Beachy, Roger N.; Yeager, Mark; Fauquet, Claude

    1998-01-01

    The translocation of rice yellow mottle virus (RYMV) within tissues of inoculated and systemically infected Oryza sativa L. leaves was characterized by Western immunoblotting, Northern blotting, and electron microscopy of thin sections. In inoculated leaves, RYMV RNA and coat protein first were detected at 3 and 5 days postinoculation, respectively. By 6 days postinoculation, RYMV had spread systemically to leaves, and virus particles were observed in most cell types, including epidermal, mesophyll, bundle sheath, and vascular parenchyma cells. Most of the virions accumulated in large crystalline patches in xylem parenchyma cells and sieve elements. Colocalization of a cell wall marker for cellulosic β-(1–4)-d-glucans and anti-RYMV antibodies over vessel pit membranes suggests a pathway for virus migration between vessels. We propose that the partial digestion of pit membranes resulting from programmed cell death may permit virus migration through them, concomitant with autolysis. In addition, displacement of the Ca2+ from pit membranes to virus particles may contribute to the disruption of the pit membranes and facilitate systemic virus transport. PMID:9501261

  4. Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line

    PubMed Central

    Dyer, Kimberly D; Schellens, Ingrid MM; Bonville, Cynthia A; Martin, Brittany V; Domachowske, Joseph B; Rosenberg, Helene F

    2007-01-01

    Pneumonia virus of mice (PVM; family Paramyxoviridae, subfamily Pneumovirinae) is a natural respiratory pathogen of rodent species and an important new model for the study of severe viral bronchiolitis and pneumonia. However, despite high virus titers typically detected in infected mouse lung tissue in vivo, cell lines used routinely for virus propagation in vitro are not highly susceptible to PVM infection. We have evaluated several rodent and primate cell lines for susceptibility to PVM infection, and detected highest virus titers from infection of the mouse monocyte-macrophage RAW 264.7 cell line. Additionally, virus replication in RAW 264.7 cells induces the synthesis and secretion of proinflammatory cytokines relevant to respiratory virus disease, including tumor necrosis factor-α (TNF-α), interferon-β (IFN-β), macrophage inflammatory proteins 1α and 1β (MIP-1α and MIP-1β) and the functional homolog of human IL-8, mouse macrophage inflammatory peptide-2 (MIP-2). Identification and characterization of a rodent cell line that supports the replication of PVM and induces the synthesis of disease-related proinflammatory mediators will facilitate studies of molecular mechanisms of viral pathogenesis that will complement and expand on findings from mouse model systems. PMID:17547763

  5. Enhanced replication of herpes simplex virus type 1 in human cells

    SciTech Connect

    Miller, C.S.; Smith, K.O. )

    1991-02-01

    The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate (MMS), methyl methanethiosulfonate (MMTS), ultraviolet light (UV), or gamma radiation (GR)) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of the infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes.

  6. Novel Cell Culture-Adapted Genotype 2a Hepatitis C Virus Infectious Clone

    PubMed Central

    Date, Tomoko; Kato, Takanobu; Kato, Junko; Takahashi, Hitoshi; Morikawa, Kenichi; Akazawa, Daisuke; Murayama, Asako; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi

    2012-01-01

    Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones. PMID:22787209

  7. Modeling HIV-1 Latency in Primary T Cells Using a Replication-Competent Virus.

    PubMed

    Martins, Laura J; Bonczkowski, Pawel; Spivak, Adam M; De Spiegelaere, Ward; Novis, Camille L; DePaula-Silva, Ana Beatriz; Malatinkova, Eva; Typsteen, Wim; Bosque, Alberto; Vanderkerckhove, Linos; Planelles, Vicente

    2016-02-01

    HIV-1 latently infected cells in vivo can be found in extremely low frequencies. Therefore, in vitro cell culture models have been used extensively for the study of HIV-1 latency. Often, these in vitro systems utilize defective viruses. Defective viruses allow for synchronized infections and circumvent the use of antiretrovirals. In addition, replication-defective viruses cause minimal cytopathicity because they fail to spread and usually do not encode env or accessory genes. On the other hand, replication-competent viruses encode all or most viral genes and better recapitulate the nuances of the viral replication cycle. The study of latency with replication-competent viruses requires the use of antiretroviral drugs in culture, and this mirrors the use of antiretroviral treatment (ART) in vivo. We describe a model that utilizes cultured central memory CD4(+) T cells and replication-competent HIV-1. This method generates latently infected cells that can be reactivated using latency reversing agents in the presence of antiretroviral drugs. We also describe a method for the removal of productively infected cells prior to viral reactivation, which takes advantage of the downregulation of CD4 by HIV-1, and the use of a GFP-encoding virus for increased throughput. PMID:26171776

  8. Enhanced natural killer-cell and T-cell responses to influenza A virus during pregnancy

    PubMed Central

    Kay, Alexander W.; Fukuyama, Julia; Aziz, Natali; Dekker, Cornelia L.; Mackey, Sally; Swan, Gary E.; Davis, Mark M.; Holmes, Susan; Blish, Catherine A.

    2014-01-01

    Pregnant women experience increased morbidity and mortality after influenza infection, for reasons that are not understood. Although some data suggest that natural killer (NK)- and T-cell responses are suppressed during pregnancy, influenza-specific responses have not been previously evaluated. Thus, we analyzed the responses of women that were pregnant (n = 21) versus those that were not (n = 29) immediately before inactivated influenza vaccination (IIV), 7 d after vaccination, and 6 wk postpartum. Expression of CD107a (a marker of cytolysis) and production of IFN-γ and macrophage inflammatory protein (MIP) 1β were assessed by flow cytometry. Pregnant women had a significantly increased percentage of NK cells producing a MIP-1β response to pH1N1 virus compared with nonpregnant women pre-IIV [median, 6.66 vs. 0.90% (P = 0.0149)] and 7 d post-IIV [median, 11.23 vs. 2.81% (P = 0.004)], indicating a heightened chemokine response in pregnant women that was further enhanced by the vaccination. Pregnant women also exhibited significantly increased T-cell production of MIP-1β and polyfunctionality in NK and T cells to pH1N1 virus pre- and post-IIV. NK- and T-cell polyfunctionality was also enhanced in pregnant women in response to the H3N2 viral strain. In contrast, pregnant women had significantly reduced NK- and T-cell responses to phorbol 12-myristate 13-acetate and ionomycin. This type of stimulation led to the conclusion that NK- and T-cell responses during pregnancy are suppressed, but clearly this conclusion is not correct relative to the more biologically relevant assays described here. Robust cellular immune responses to influenza during pregnancy could drive pulmonary inflammation, explaining increased morbidity and mortality. PMID:25246558

  9. Quantitation, in vitro propagation, and characterization of preleukemic cells induced by radiation leukemia virus

    SciTech Connect

    Yefenof, E.; Epszteyn, S.; Kotler, M. )

    1991-04-15

    Intrathymic (i.t.) inoculation of radiation leukemia virus into C57BL/6 mice induces a population of preleukemic (PL) cells that can progress into mature thymic lymphomas upon transfer into syngeneic recipients. A minimum of 10(3) PL thymic cells are required to induce lymphomas in the recipient. Most of the individual lymphomas developed in mice which were inoculated with cells of a single PL thymus, derived from different T-cell precursors. PL thymic cells could be grown in vitro on a feeder layer consisting of splenic stromal cells. Growth medium was supplemented with supernatant harvested from an established radiation leukemia virus-induced lymphoma cell line (SR4). The in vitro-grown PL cells were characterized as Thy-1+, CD4+, CD8- T-cells, most of which expressed radiation leukemia virus antigens. Cultured PL cells were found to be nontumorigenic, based on their inability to form s.c. tumors. However, these cells could develop into thymic lymphomas if inoculated i.t. into syngeneic recipients. A culture of PL cells, maintained for 2 mo, showed clonal T-cell receptor arrangement. Lymphomas which developed in several recipient mice upon injection with these PL cells were found to possess the same T-cell receptor arrangement. These results indicate that PL cells can be adapted for in vitro growth while maintaining their preleukemic character.

  10. Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection

    PubMed Central

    Lang, P A; Meryk, A; Pandyra, A A; Brenner, D; Brüstle, A; Xu, H C; Merches, K; Lang, F; Khairnar, V; Sharma, P; Funkner, P; Recher, M; Shaabani, N; Duncan, G S; Duhan, V; Homey, B; Ohashi, P S; Häussinger, D; Knolle, P A; Honke, N; Mak, T W; Lang, K S

    2015-01-01

    During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8+ T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso–/–) mice reduced CD8+ T-cell function in the liver and resulted in virus persistence. Furthermore, Toso–/– DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection. PMID:25257173

  11. Toso regulates differentiation and activation of inflammatory dendritic cells during persistence-prone virus infection.

    PubMed

    Lang, P A; Meryk, A; Pandyra, A A; Brenner, D; Brüstle, A; Xu, H C; Merches, K; Lang, F; Khairnar, V; Sharma, P; Funkner, P; Recher, M; Shaabani, N; Duncan, G S; Duhan, V; Homey, B; Ohashi, P S; Häussinger, D; Knolle, P A; Honke, N; Mak, T W; Lang, K S

    2015-01-01

    During virus infection and autoimmune disease, inflammatory dendritic cells (iDCs) differentiate from blood monocytes and infiltrate infected tissue. Following acute infection with hepatotropic viruses, iDCs are essential for re-stimulating virus-specific CD8(+) T cells and therefore contribute to virus control. Here we used the lymphocytic choriomeningitis virus (LCMV) model system to identify novel signals, which influence the recruitment and activation of iDCs in the liver. We observed that intrinsic expression of Toso (Faim3, FcμR) influenced the differentiation and activation of iDCs in vivo and DCs in vitro. Lack of iDCs in Toso-deficient (Toso(-/-)) mice reduced CD8(+) T-cell function in the liver and resulted in virus persistence. Furthermore, Toso(-/-) DCs failed to induce autoimmune diabetes in the rat insulin promoter-glycoprotein (RIP-GP) autoimmune diabetes model. In conclusion, we found that Toso has an essential role in the differentiation and maturation of iDCs, a process that is required for the control of persistence-prone virus infection. PMID:25257173

  12. Melaleuca alternifolia concentrate inhibits in vitro entry of influenza virus into host cells.

    PubMed

    Li, Xinghua; Duan, Songwei; Chu, Cordia; Xu, Jun; Zeng, Gucheng; Lam, Alfred King-Yin; Zhou, Junmei; Yin, Yue; Fang, Danyun; Reynolds, Maxwell John; Gu, Huaiyu; Jiang, Lifang

    2013-01-01

    Influenza virus causes high morbidity among the infected population annually and occasionally the spread of pandemics. Melaleuca alternifolia Concentrate (MAC) is an essential oil derived from a native Australian tea tree. Our aim was to investigate whether MAC has any in vitro inhibitory effect on influenza virus infection and what mechanism does the MAC use to fight the virus infection. In this study, the antiviral activity of MAC was examined by its inhibition of cytopathic effects. In silico prediction was performed to evaluate the interaction between MAC and the viral haemagglutinin. We found that when the influenza virus was incubated with 0.010% MAC for one hour, no cytopathic effect on MDCK cells was found after the virus infection and no immunofluorescence signal was detected in the host cells. Electron microscopy showed that the virus treated with MAC retained its structural integrity. By computational simulations, we found that terpinen-4-ol, which is the major bioactive component of MAC, could combine with the membrane fusion site of haemagglutinin. Thus, we proved that MAC could prevent influenza virus from entering the host cells by disturbing the normal viral membrane fusion procedure. PMID:23966077

  13. Lower Virus Infections in Varroa destructor-Infested and Uninfested Brood and Adult Honey Bees (Apis mellifera) of a Low Mite Population Growth Colony Compared to a High Mite Population Growth Colony

    PubMed Central

    Emsen, Berna; Hamiduzzaman, Mollah Md.; Goodwin, Paul H.; Guzman-Novoa, Ernesto

    2015-01-01

    A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection. PMID:25723540

  14. Lower virus infections in Varroa destructor-infested and uninfested brood and adult honey bees (Apis mellifera) of a low mite population growth colony compared to a high mite population growth colony.

    PubMed

    Emsen, Berna; Hamiduzzaman, Mollah Md; Goodwin, Paul H; Guzman-Novoa, Ernesto

    2015-01-01

    A comparison was made of the prevalence and relative quantification of deformed wing virus (DWV), Israeli acute paralysis virus (IAPV), black queen cell virus (BQCV), Kashmir bee virus (KBV), acute bee paralysis virus (ABPV) and sac brood virus (SBV) in brood and adult honey bees (Apis mellifera) from colonies selected for high (HMP) and low (LMP) Varroa destructor mite population growth. Two viruses, ABPV and SBV, were never detected. For adults without mite infestation, DWV, IAPV, BQCV and KBV were detected in the HMP colony; however, only BQCV was detected in the LMP colony but at similar levels as in the HMP colony. With mite infestation, the four viruses were detected in adults of the HMP colony but all at higher amounts than in the LMP colony. For brood without mite infestation, DWV and IAPV were detected in the HMP colony, but no viruses were detected in the LMP colony. With mite infestation of brood, the four viruses were detected in the HMP colony, but only DWV and IAPV were detected and at lower amounts in the LMP colony. An epidemiological explanation for these results is that pre-experiment differences in virus presence and levels existed between the HMP and LMP colonies. It is also possible that low V. destructor population growth in the LMP colony resulted in the bees being less exposed to the mite and thus less likely to have virus infections. LMP and HMP bees may have also differed in susceptibility to virus infection. PMID:25723540

  15. Generation of Influenza Virus from Avian Cells Infected by Salmonella Carrying the Viral Genome

    PubMed Central

    Zhang, Xiangmin; Kong, Wei; Wanda, Soo-Young; Xin, Wei; Alamuri, Praveen; Curtiss, Roy

    2015-01-01

    Domestic poultry serve as intermediates for transmission of influenza A virus from the wild aquatic bird reservoir to humans, resulting in influenza outbreaks in poultry and potential epidemics/pandemics among human beings. To combat emerging avian influenza virus, an inexpensive, heat-stable, and orally administered influenza vaccine would be useful to vaccinate large commercial poultry flocks and even migratory birds. Our hypothesized vaccine is a recombinant attenuated bacterial strain able to mediate production of attenuated influenza virus in vivo to induce protective immunity against influenza. Here we report the feasibility and technical limitations toward such an ideal vaccine based on our exploratory study. Five 8-unit plasmids carrying a chloramphenicol resistance gene or free of an antibiotic resistance marker were constructed. Influenza virus was successfully generated in avian cells transfected by each of the plasmids. The Salmonella carrier was engineered to allow stable maintenance and conditional release of the 8-unit plasmid into the avian cells for recovery of influenza virus. Influenza A virus up to 107 50% tissue culture infective doses (TCID50)/ml were recovered from 11 out of 26 co-cultures of chicken embryonic fibroblasts (CEF) and Madin-Darby canine kidney (MDCK) cells upon infection by the recombinant Salmonella carrying the 8-unit plasmid. Our data prove that a bacterial carrier can mediate generation of influenza virus by delivering its DNA cargoes into permissive host cells. Although we have made progress in developing this Salmonella influenza virus vaccine delivery system, further improvements are necessary to achieve efficient virus production, especially in vivo. PMID:25742162

  16. Immune response and resistance to Rous sarcoma virus challenge of chickens immunized with cell-associated glycoproteins provided with a recombinant avian leukosis virus.

    PubMed Central

    Chebloune, Y; Rulka, J; Cosset, F L; Valsesia, S; Ronfort, C; Legras, C; Drynda, A; Kuzmak, J; Nigon, V M; Verdier, G

    1991-01-01

    The Rous-associated virus 1 env gene, which encodes the envelope gp85 and gp37 glycoproteins, was isolated and inserted in place of the v-erbB oncogene into an avian erythroblastosis virus-based vector, carrying the neo resistance gene substituted for the v-erbA oncogene, to generate the pNEA recombinant vector. A helper-free virus stock of the pNEA vector was produced on an avian transcomplementing cell line and used to infect primary chicken embryo fibroblasts (CEFs) or quail QT6 cells. These infected cells, selected with G418 (CEF/NEA and QT6/NEA, respectively) were found to be resistant to superinfections with subgroup A retroviruses. The CEF/NEA preparations were used as a cell-associated antigen to inoculate adult chickens by the intravenous route compared with direct inoculations of NEA recombinant helper-free virus used as a cell-free antigen. Chickens injected with the cell-associated antigen (CEF/NEA) exhibited an immune response demonstrated by induction of high titers of neutralizing antibodies and were found to be protected against tumor production after Rous sarcoma virus A challenge. Conversely, no immune response and no protection against Rous sarcoma virus A challenge were observed in chickens directly inoculated with cell-free NEA recombinant virus or in sham-inoculated chickens. PMID:1654445

  17. Macrophages as target cells for Mayaro virus infection: involvement of reactive oxygen species in the inflammatory response during virus replication.

    PubMed

    Cavalheiro, Mariana G; Costa, Leandro Silva DA; Campos, Holmes S; Alves, Letícia S; Assunção-Miranda, Iranaia; Poian, Andrea T DA

    2016-09-01

    Alphaviruses among the viruses that cause arthritis, consisting in a public health problem worldwide by causing localized outbreaks, as well as large epidemics in humans. Interestingly, while the Old World alphaviruses are arthritogenic, the New World alphaviruses cause encephalitis. One exception is Mayaro virus (MAYV), which circulates exclusively in South America but causes arthralgia and is phylogenetically related to the Old World alphaviruses. Although MAYV-induced arthritis in humans is well documented, the molecular and cellular factors that contribute to its pathogenesis are completely unknown. In this study, we demonstrated for the first time that macrophages, key players in arthritis development, are target cells for MAYV infection, which leads to cell death through apoptosis. We showed that MAYV replication in macrophage induced the expression of TNF, a cytokine that would contribute to pathogenesis of MAYV fever, since TNF promotes an inflammatory profile characteristic of arthritis. We also found a significant increase in the production of reactive oxygen species (ROS) at early times of infection, which coincides with the peak of virus replication and precedes TNF secretion. Treatment of the cells with antioxidant agents just after infection completely abolished TNF secretion, indicating an involvement of ROS in inflammation induced during MAYV infection. PMID:27627069

  18. Occurrence of Deformed wing virus, Chronic bee paralysis virus and mtDNA variants in haplotype K of Varroa destructor mites in Syrian apiaries.

    PubMed

    Elbeaino, Toufic; Daher-Hjaij, Nouraldin; Ismaeil, Faiz; Mando, Jamal; Khaled, Bassem Solaiman; Kubaa, Raied Abou

    2016-05-01

    A small-scale survey was conducted on 64 beehives located in four governorates of Syria in order to assess for the first time the presence of honeybee-infecting viruses and of Varroa destructor mites in the country. RT-PCR assays conducted on 192 honeybees (Apis mellifera L.) using virus-specific primers showed that Deformed wing virus (DWV) was present in 49 (25.5%) of the tested samples and Chronic bee paralysis virus (CBPV) in 2 (1.04%), whereas Acute bee paralysis virus, Sacbrood virus, Black queen cell virus and Kashmir bee virus were absent. Nucleotide sequences of PCR amplicons obtained from DWV and CBPV genomes shared 95-97 and 100% identity with isolates reported in the GenBank, respectively. The phylogenetic tree grouped the Syrian DWV isolates in one cluster, distinct from all those of different origins reported in the database. Furthermore, 19 adult V. destructor females were genetically analyzed by amplifying and sequencing four fragments in cytochrome oxidase subunit 1 (cox1), ATP synthase 6 (atp6), cox3 and cytochrome b (cytb) mitochondrial DNA (mtDNA) genes. Sequences of concatenated V. destructor mtDNA genes (2696 bp) from Syria were similar to the Korean (K) haplotype and were found recurrently in all governorates. In addition, two genetic lineages of haplotype K with slight variations (0.2-0.3%) were present only in Tartous and Al-Qunaitra governorates. PMID:26914360

  19. How many gamergates is an ant queen worth?

    NASA Astrophysics Data System (ADS)

    Monnin, Thibaud; Peeters, Christian

    2008-02-01

    Ant reproductives exhibit different morphological adaptations linked to dispersal and fertility. By reviewing the literature on taxa where workers can reproduce sexually (i.e. become gamergates) we show that (1) species with a single gamergate generally have lost the winged queen caste, whereas only half of the species with several gamergates have, and (2) single-gamergate species have smaller colonies than multiple-gamergate species. Comparison with “classical” ants without gamergates, where having one vs having several winged queens are two distinct syndromes, suggests that having one vs having several gamergates are not. Gamergate number does not affect the success of colony fission, but retention of the queen caste permits the option of independent foundation.

  20. Genetic studies of cell fusion induced by herpes simplex virus type 1

    SciTech Connect

    Read, G.S.; Person, S.; Keller, P.M.

    1980-07-01

    Eight cell fusion-causing syn mutants were isolated from the KOS strain of herpes simplex virus type 1. Unlike the wild-type virus, the mutants produced plaques containing multinucleated cells, or syncytia. Fusion kinetics curves were established with a Coulter Counter assay for the mutants and wild-type virus in single infections of human embryonic lung (HEL) cells, for the mutants and wild-type virus in mixed infections (dominance test), and for pairs of mutants in mixed infection and proceeded with an exponential decrease in the number of small single cells. At some later time that was characteristic of the mutant, there was a significant reduction in the rate of fusion for all but possibly one of the mutants. Although the wild-type virus did not produce syncytial plaques, it did induce a small amount of fusion that stopped abruptly about 2 h after it started. These data are consistent with the hypothesis that both mutants and wild type induce an active fusion inducer and that the activity of this inducer is subsequently inhibited. The extent of fusion is apparently determined by the length of the interval during which the fusion inducer is active. That fusion is actively inhibited in wild-type infections is indicated by the observation that syn mutant-infected cells fused more readily with uninfected cells than with wild type-infected cells.

  1. Replication of type I herpes simplex virus in primary cultures of hairy cell leukemic leukocytes.

    PubMed Central

    Pozner, L. H.; Daniels, C. A.; Cooper, J. A.; Cohen, H. J.; Logue, G. L.; Croker, B. P.

    1978-01-01

    The ability of leukemic leukocytes to support the replication of herpes simplex virus (HSV) was studied. Mononuclear leukocytes (MNL) from the peripheral blood of patients with a variety of lymphoid leukemias were isolated on Ficoll-Hypaque gradients and infected with HSV at a multiplicity of infection of 5 to 10. No virus growth was detected in cells from patients with chronic lymphocytic leukemia (9), acute lymphocytic leukemia (1), or lymphosarcoma cell leukemia (2), HSV replication did occur in hairy cell leukemic MNL from all of 4 patients studied. Maximal titers of 10(3.7) to 10(4.7) PFU/ml occurred 1 to 7 days after incubation. By electron microscopy, herpesvirus particles were seen in the nuclei of these infected cells after 3 days of culture, but none was seen in the cells not exposed to virus. Fluorescent antibody examination confirmed the presence of HSV antigens in the nuclei of infected hairy cells. No difference in the adsorption or penetration of the virus was found with the various MNL studied. Productive infection of the cells thus appeared to depend on the ability of the leukocyte ;o support a later stage of infection, either uncoating or replication of the virus. Images Figure 1 PMID:202167

  2. Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry.

    PubMed

    Pohl, Marie O; Stertz, Silke

    2015-01-01

    Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV. PMID:26575457

  3. Derivation of cell-adapted Sacbrood virus (SBV) from the native Korean honeybee.

    PubMed

    Kweon, Chang-Hee; Yoo, Mi-Sun; Noh, Jin-Hyeong; Reddy, Kondreddy Eswar; Yang, Dong-Kun; Cha, Sang-Ho; Kang, Seung-Won

    2015-02-16

    Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of SBV in cell cultures. The replication signs of KSBV after passages from mammalian cells was identified and confirmed by using combined approaches with nested, quantitative, negative-strand PCR and electron microscopy along with in vivo experiment. The results revealed that mammalian cell lines, including Vero cells could support the replication KSBV. Although there were no signs of cytopathic effect (CPE) in cells, it was for the first time demonstrated that SBV could be replicated in cells through the sequential passages linked with cell adaptation. KSBV from the present study would be a valuable source to understand the mechanism of pathogenicity of sacbrood virus in the future. PMID:25527463

  4. Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors

    SciTech Connect

    Filone, Claire Marie; Heise, Mark; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu; Bertolotti-Ciarlet, Andrea . E-mail: aciarlet@mail.med.upenn.edu

    2006-12-20

    Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell-cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by {beta}-galactosidase {alpha}-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell-cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell-cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.

  5. Dose-Dependent Changes in Influenza Virus-Infected Dendritic Cells Result in Increased Allogeneic T-Cell Proliferation at Low, but Not High, Doses of Virus

    PubMed Central

    Oh, SangKon; McCaffery, J. Michael; Eichelberger, Maryna C.

    2000-01-01

    During the acute phase of infection with influenza A virus, the degree of lymphopenia correlates with severity of disease. Factors that contribute to T-cell activation during influenza virus infection may contribute to this observation. Since the immune response is initiated when dendritic cells (DC) interact with T cells, we have established an in vitro system to examine the effects of influenza virus infection on DC function. Our results show that allogeneic T-cell proliferation was dependent on the dose of A/PR/8/34 used to infect DC, with enhanced responses at low, but not high, multiplicities of infection. The lack of enhancement at high virus doses was not primarily due to the increased rate of DC apoptosis, but required viral replication and neuraminidase (NA) activity. Clusters that formed between DC or between DC and T cells were also dependent on the viral dose. This change in cellular interaction may oppose T-cell proliferation in response to DC infected with high doses of PR8, since the increased contact between DC resulted in the exclusion of T cells. The enhanced alloreactive T-cell response was restored by neutralization of transforming growth factor β1 (TGF-β1). It is likely that NA present on viral particles released from DC infected with high doses of PR8 activates TGF-β1. Future studies will determine the mechanism by which TGF-β1 modifies the in vitro T-cell response and address the contribution of this cytokine to the lymphopenia observed in severe disease. PMID:10823850

  6. Envelope Glycoprotein Internalization Protects Human and Simian Immunodeficiency Virus-Infected Cells from Antibody-Dependent Cell-Mediated Cytotoxicity

    PubMed Central

    von Bredow, Benjamin; Arias, Juan F.; Heyer, Lisa N.; Gardner, Matthew R.; Farzan, Michael; Rakasz, Eva G.

    2015-01-01

    ABSTRACT The cytoplasmic tails of human and simian immunodeficiency virus (HIV and SIV, respectively) envelope glycoproteins contain a highly conserved, membrane-proximal endocytosis motif that prevents the accumulation of Env on the surface of infected cells prior to virus assembly. Using an assay designed to measure the killing of virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC), we show that substitutions in this motif increase the susceptibility of HIV-1- and SIV-infected cells to ADCC in a manner that directly correlates with elevated Env levels on the surface of virus-infected cells. In the case of HIV-1, this effect is additive with a deletion in vpu recently shown to enhance the susceptibility of HIV-1-infected cells to ADCC as a result of tetherin-mediated retention of budding virions on the cell surface. These results reveal a previously unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from antibody responses by regulating the amount of Env present on the cell surface. IMPORTANCE This study reveals an unappreciated role for the membrane-proximal endocytosis motif of gp41 in protecting HIV-1- and SIV-infected cells from elimination by Env-specific antibodies. Thus, strategies designed to interfere with this mechanism of Env internalization may improve the efficacy of antibody-based vaccines and antiretroviral therapies designed to enhance the immunological control of HIV-1 replication in chronically infected individuals. PMID:26269175

  7. Quantitative analysis of human immunodeficiency virus type 1-infected CD4(+) cell proteome: Dysregulated cell cycle progression and nuclear transport coincide with robust virus production

    SciTech Connect

    Chan, Eric Y.; Qian, Weijun; Diamond, Deborah L.; Liu, Tao; Gritsenko, Marina A.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Katze, Michael G.

    2007-07-01

    Relatively little is known at the functional genomic level about the global host response to HIV-1 infection. Microarray analyses by several laboratories, including our own, have revealed that human immunodeficiency virus type 1 infection causes significant changes in host mRNA abundance and regulation of several cellular biological pathways. However, it remains unclear what consequences these changes bring about at the protein level. Here we report the expression levels of ~3,200 proteins assessed in the CD4+ CEMx174 cell line after infection with HIV-1 LAI, using liquid chromatography-mass spectrometry coupled with stable isotope labeling and the accurate mass and time (AMT) tag approach. Further, we found that 687 (21%) proteins changed in abundance at the peak of virus production at 36h post-infection. Pathway analysis revealed that the differential expression of proteins were concentrated in select biological pathways, exemplified by ubiquitin conjugating enzymes in the ubiquitination, carrier proteins in nucleo-cytoplasmic transport, cyclin-dependent kinase in cell cycle progression, and pyruvate dehydrogenase of the citrate cycle. Moreover, we observed changes in the abundance of proteins with known interactions with HIV-1 viral proteins. Our proteomic analysis captured changes in the host protein milieu at the time of robust virus production, accompanied by a moderate accumulation of G1/G0-phase cells. We will discuss the contributions of these changes to virus production in the infected cells.

  8. 76 FR 2438 - Culturally Significant Objects Imported for Exhibition Determinations: “Kings, Queens, and...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-01-13

    ... the exhibition ``Kings, Queens, and Courtiers: Art in Early Renaissance France'' imported from abroad... Culturally Significant Objects Imported for Exhibition Determinations: ``Kings, Queens, and Courtiers: Art in Early Renaissance France'' SUMMARY: Notice is hereby given of the following determinations: Pursuant...

  9. Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

    PubMed Central

    Choi, In Young; Lim, HoTae; Lee, Gabsang

    2014-01-01

    A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases. Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner. PMID:24798302

  10. Equine Endothelial Cells Support Productive Infection of Equine Infectious Anemia Virus

    PubMed Central

    Maury, Wendy; Oaks, J. Lindsay; Bradley, Sarahann

    1998-01-01

    Previous cell infectivity studies have demonstrated that the lentivirus equine infectious anemia virus (EIAV) infects tissue macrophages in vivo and in vitro. In addition, some strains of EIAV replicate to high titer in vitro in equine fibroblasts and fibroblast cell lines. Here we report a new cell type, macrovascular endothelial cells, that is infectible with EIAV. We tested the ability of EIAV to infect purified endothelial cells isolated from equine umbilical cords and renal arteries. Infectivity was detected by cell supernatant reverse transcriptase positivity, EIAV antigen positivity within individual cells, and the detection of viral RNA by in situ hybridization. Virus could rapidly spread through the endothelial cultures, and the supernatants of infected cultures contained high titers of infectious virus. There was no demonstrable cell killing in infected cultures. Three of four strains of EIAV that were tested replicated in these cultures, including MA-1, a fibroblast-tropic strain, Th.1, a macrophage-tropic strain, and WSU5, a strain that is fibroblast tropic and can cause disease. Finally, upon necropsy of a WSU5-infected horse 4 years postinfection, EIAV-positive endothelial cells were detected in outgrowths of renal artery cultures. These findings identify a new cell type that is infectible with EIAV. The role of endothelial cell infection in the course of equine infectious anemia is currently unknown, but endothelial cell infection may be involved in the edema that can be associated with infection. Furthermore, the ability of EIAV to persistently infect endothelial cultures and the presence of virus in endothelial cells from a long-term carrier suggest that this cell type can serve as a reservoir for the virus during subclinical phases of infection. PMID:9765477

  11. Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney

    PubMed Central

    Murayama, Asako; Sugiyama, Nao; Wakita, Takaji

    2016-01-01

    ABSTRACT A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells. PMID:27302754

  12. Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

    PubMed Central

    Kohl, S; Drath, D B; Loo, L S

    1982-01-01

    Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host. PMID:6295943

  13. Murine cellular cytotoxicity to syngeneic and xenogeneic herpes simplex virus-infected cells.

    PubMed

    Kohl, S; Drath, D B; Loo, L S

    1982-12-01

    Cellular cytotoxicity of C57BL/6 adult mice peritoneal cells to xenogeneic (Chang liver) and syngeneic (BL/6-WT3) herpes simplex virus (HSV)-infected cells was analyzed in a 6-h 51Cr release assay. There was no difference in antibody-dependent cellular cytotoxicity to either target. There was no natural killer cytotoxicity to targets with cells from uninfected mice except at very high effector cell ratios. HSV-infected (2 X 10(4) PFU intraperitoneally 1 day previously) mice mediated significantly higher antibody-dependent cellular cytotoxicity and required less antibody (10(-5) versus 10(-2) dilution), fewer cells, and less time to kill than cells from uninfected mice. HSV-infected mice mediated natural killer cytotoxicity but preferentially killed syngeneic HSV-infected cells. Stimulation of cytotoxicity was not virus specific since influenza-infected mice mediated similar levels of cytotoxicity to HSV-infected targets. There was no difference in morphology (95% macrophage) or in the percentage of FcR-positive cells, but infected mice had more peritoneal cells and generated higher levels of superoxide in response to opsonized zymosan or phorbolmyristate acetate. These data demonstrate nonspecific virus-stimulated metabolic and effector cell function which may enhance clearance of virus in an infected host. PMID:6295943

  14. Replication of parainfluenza (Sendai) virus in isolated rat pulmonary type II alveolar epithelial cells.

    PubMed Central

    Castleman, W. L.; Northrop, P. J.; McAllister, P. K.

    1989-01-01

    The major objectives of this study were to determine whether alveolar type II epithelial cells isolated from rat lung and maintained in tissue culture would support productive replication of parainfluenza type 1 (Sendai) virus and to determine whether isolated type II cells from neonatal (5-day-old) rats that are more susceptible to viral-induced alveolar dysplasia supported viral replication to a greater extent than those from weanling (25-day-old) rats. Isolated and cultured type II cells from neonatal and weanling rats that were inoculated with Sendai virus supported productive replication as indicated by ultrastructural identification of budding virions and viral nucleocapsids in type II cells and by demonstration of rising titers of infectious virus from inoculated type II cell cultures. Alveolar macrophages from neonatal and weanling rats also supported viral replication, although infectious viral titers in macrophage cultures were lower than those from type II cell cultures. Only minor differences were detected between viral titers from neonatal and weanling type II epithelial cell cultures. Higher densities of viral nucleocapsids were observed in neonatal type II cells than in those from weanling rats. The results indicate that isolated type II alveolar epithelial cells support productive replication of parainfluenza virus and that type II cells are probably more efficient in supporting productive viral replication than are alveolar macrophages. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:2541612

  15. Comparative analysis of signature genes in porcine reproductive and respiratory syndrome virus (PRRSV)-infected porcine monocyte-derived dendritic cells at differential activation statuses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Activation statuses of monocytic cells, e.g. monocytes, macrophages and dendritic cells (DCs), are critically important for antiviral immunity. In particular, some devastating viruses, including porcine reproductive and respiratory syndrome virus (PRRSV), are capable of directly infecting these cell...

  16. Pinpointing retrovirus entry sites in cells expressing alternatively spliced receptor isoforms by single virus imaging

    PubMed Central

    2014-01-01

    Background The majority of viruses enter host cells via endocytosis. Current knowledge of viral entry pathways is largely based upon infectivity measurements following genetic and/or pharmacological interventions that disrupt vesicular trafficking and maturation. Imaging of single virus entry in living cells provides a powerful means to delineate viral trafficking pathways and entry sites under physiological conditions. Results Here, we visualized single avian retrovirus co-trafficking with markers for early (Rab5) and late (Rab7) endosomes, acidification of endosomal lumen and the resulting viral fusion measured by the viral content release into the cytoplasm. Virus-carrying vesicles either merged with the existing Rab5-positive early endosomes or slowly accumulated Rab5. The Rab5 recruitment to virus-carrying endosomes correlated with acidification of their lumen. Viral fusion occurred either in early (Rab5-positive) or intermediate (Rab5- and Rab7-positive) compartments. Interestingly, different isoforms of the cognate receptor directed virus entry from distinct endosomes. In cells expressing the transmembrane receptor, viruses preferentially entered and fused with slowly maturing early endosomes prior to accumulation of Rab7. By comparison, in cells expressing the GPI-anchored receptor, viruses entered both slowly and quickly maturing endosomes and fused with early (Rab5-positive) and intermediate (Rab5- and Rab7-positive) compartments. Conclusions Since the rate of low pH-triggered fusion was independent of the receptor isoform, we concluded that the sites of virus entry are determined by the kinetic competition between endosome maturation and viral fusion. Our findings demonstrate the ability of this retrovirus to enter cells via alternative endocytic pathways and establish infection by releasing its content from distinct endosomal compartments. PMID:24935247

  17. Studies on Sam68 a cell factor involved in the life cycle of foot-and-mouth disease virus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As with other RNA viruses, Foot-and-Mouth Disease Virus (FMDV) recruits various host cell factors to assist in translation and replication of the virus genome. While FMDV translation has been thoroughly investigated, much remains unknown regarding replication of the positive-sense RNA genome. In th...

  18. Hepatitis E Virus Produced from Cell Culture Has a Lipid Envelope.

    PubMed

    Qi, Ying; Zhang, Feng; Zhang, Li; Harrison, Tim J; Huang, Weijin; Zhao, Chenyan; Kong, Wei; Jiang, Chunlai; Wang, Youchun

    2015-01-01

    The absence of a productive cell culture system hampered detailed analysis of the structure and protein composition of the hepatitis E virion. In this study, hepatitis E virus from a robust HEV cell culture system and from the feces of infected monkeys at the peak of virus excretion was purified by ultra-centrifugation. The common feature of the two samples after ultracentrifugation was that the ORF2 protein mainly remained in the top fractions. The ORF2 protein from cell culture system was glycosylated, with an apparent molecular weight of 88 kDa, and was not infectious in PLC/PRF/5 cells. The ORF2 protein in this fraction can bind to and protect HEV RNA from digestion by RNase A. The RNA-ORF2 product has a similar sedimentation coefficient to the virus from feces. The viral RNA in the cell culture supernatant was mainly in the fraction of 1.15 g/cm3 but that from the feces was mainly in the fraction of 1.21 g/cm3. Both were infectious in PLC/PRF/5 cells. And the fraction in the middle of the gradient (1.06 g/cm3) from the cell culture supernatant,but not that from the feces, also has ORF2 protein and HEV RNA but was not infectious in PLC/PRF/5.The infectious RNA-rich fraction from the cell culture contained ORF3 protein and lipid but the corresponding fraction from feces had no lipid and little ORF3 protein. The lipid on the surface of the virus has no effect on its binding to cells but the ORF3 protein interferes with binding. The result suggests that most of the secreted ORF2 protein is not associated with HEV RNA and that hepatitis E virus produced in cell culture differs in structure from the virus found in feces in that it has a lipid envelope. PMID:26161670

  19. Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

    PubMed Central

    Mangala Prasad, Vidya; Wang, Cheng-I; Akahata, Wataru; Ng, Lisa F. P.

    2015-01-01

    ABSTRACT Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. PMID:26537684

  20. High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara.

    PubMed

    Hebben, Matthias; Brants, Jan; Birck, Catherine; Samama, Jean-Pierre; Wasylyk, Bohdan; Spehner, Danièle; Pradeau, Karine; Domi, Arban; Moss, Bernard; Schultz, Patrick; Drillien, Robert

    2007-12-01

    Vaccinia virus vectors are attractive tools to direct high level protein synthesis in mammalian cells. In one of the most efficient strategies developed so far, the gene to be expressed is positioned downstream of a bacteriophage T7 promoter within the vaccinia genome and transcribed by the T7 RNA polymerase, also encoded by the vaccinia virus genome. Tight regulation of transcription and efficient translation are ensured by control elements of the Escherichia coli lactose operon and the encephalomyocarditis virus leader sequence, respectively. We have integrated such a stringently controlled expression system, previously used successfully in a standard vaccinia virus backbone, into the modified vaccinia virus Ankara strain (MVA). In this manner, proteins of interest can be produced in mammalian cells under standard laboratory conditions because of the inherent safety of the MVA strain. Using this system for expression of beta-galactosidase, about 15 mg protein could be produced from 10(8) BHK21 cells over a 24-h period, a value 4-fold higher than the amount produced from an identical expression system based on a standard vaccinia virus strain. In another application, we employed the MVA vector to produce human tubulin tyrosine ligase and demonstrate that this protein becomes a major cellular protein upon induction conditions and displays its characteristic enzymatic activity. The MVA vector should prove useful for many other applications in which mammalian cells are required for protein production. PMID:17892951

  1. Development and qualification of a novel virus removal filter for cell culture applications.

    PubMed

    Liu, S; Carroll, M; Iverson, R; Valera, C; Vennari, J; Turco, K; Piper, R; Kiss, R; Lutz, H

    2000-01-01

    Commercial bioreactors employing mammalian cell cultures to express biological or pharmaceutical products can become contaminated with adventitious viruses. The high expense of such a contamination can be reduced by passing all gases and fluids feeding the bioreactor through virus inactivation or removal steps, which act as viral barriers around the bioreactor. A novel virus barrier filter has been developed for removing viruses from serum-free cell culture media. This filter removes the 20 nm minute virus of mice by >3 log reduction value (LRV), the 28 nm bacteriophage PhiX174 by >4.5 LRV, the mycoplasma Acholeplasma laidlawii by > or =8.8 LRV, and the bacteria Brevundimonas diminuta by > or =9.2 LRV. Robust removal occurs primarily by size exclusion as demonstrated over a wide range of feedstocks and operating conditions. The filtered media are indistinguishable from unfiltered media in growth of cells to high densities, maintenance of cell viability, and productivity in expressing protein product. Insulin and transferrin show high passage through the filter. The virus barrier filter can be autoclaved. The relatively high membrane permeability enables the use of a moderate filtration area. PMID:10835245

  2. Dendritic Cell Internalization of Foot-and-Mouth Disease Virus: Influence of Heparan Sulfate Binding on Virus Uptake and Induction of the Immune Response▿

    PubMed Central

    Harwood, Lisa J.; Gerber, Heidi; Sobrino, Francisco; Summerfield, Artur; McCullough, Kenneth C.

    2008-01-01

    Dendritic cells (DC), which are essential for inducing and regulating immune defenses and responses, represent the critical target for vaccines against pathogens such as foot-and-mouth disease virus (FMDV). Although it is clear that FMDV enters epithelial cells via integrins, little is known about FMDV interaction with DC. Accordingly, DC internalization of FMDV antigen was analyzed by comparing vaccine virus dominated by heparan sulfate (HS)-binding variants with FMDV lacking HS-binding capacity. The internalization was most efficient with the HS-binding virus, employing diverse endocytic pathways. Moreover, internalization relied primarily on HS binding. Uptake of non-HS-binding virus by DC was considerably less efficient, so much so that it was often difficult to detect virus interacting with the DC. The HS-binding FMDV replicated in DC, albeit transiently, which was demonstrable by its sensitivity to cycloheximide treatment and the short duration of infectious virus production. There was no evidence that the non-HS-binding virus replicated in the DC. These observations on virus replication may be explained by the activities of viral RNA in the DC. When DC were transfected with infectious RNA, only 1% of the translated viral proteins were detected. Nevertheless, the transfected cells, and DC which had internalized live virus, did present antigen to lymphocytes, inducing an FMDV-specific immunoglobulin G response. These results demonstrate that DC internalization of FMDV is most efficient for vaccine virus with HS-binding capacity, but HS binding is not an exclusive requirement. Both non-HS-binding virus and infectious RNA interacting with DC induce specific immune responses, albeit less efficiently than HS-binding virus. PMID:18448534

  3. African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

    PubMed Central

    Nogal, María L.; González de Buitrago, Gonzalo; Rodríguez, Clara; Cubelos, Beatriz; Carrascosa, Angel L.; Salas, María L.; Revilla, Yolanda

    2001-01-01

    African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis. PMID:11222676

  4. A thiopurine drug inhibits West Nile virus production in cell culture, but not in mice.

    PubMed

    Lim, Pei-Yin; Keating, Julie A; Hoover, Spencer; Striker, Rob; Bernard, Kristen A

    2011-01-01

    Many viruses within the Flavivirus genus cause significant disease in humans; however, effective antivirals against these viruses are not currently available. We have previously shown that a thiopurine drug, 6-methylmercaptopurine riboside (6MMPr), inhibits replication of distantly related viruses within the Flaviviridae family in cell culture, including bovine viral diarrhea virus and hepatitis C virus replicon. Here we further examined the potential antiviral effect of 6MMPr on several diverse flaviviruses. In cell culture, 6MMPr inhibited virus production of yellow fever virus, dengue virus-2 (DENV-2) and West Nile virus (WNV) in a dose-dependent manner, and DENV-2 was significantly more sensitive to 6MMPr treatment than WNV. We then explored the use of 6MMPr as an antiviral against WNV in an immunocompetent mouse model. Once a day treatment of mice with 0.5 mg 6MMPr was just below the toxic dose in our mouse model, and this dose was used in subsequent studies. Mice were treated with 6MMPr immediately after subcutaneous inoculation with WNV for eight consecutive days. Treatment with 6MMPr exacerbated weight loss in WNV-inoculated mice and did not significantly affect mortality. We hypothesized that 6MMPr has low bioavailability in the central nervous system (CNS) and examined the effect of pre-treatment with 6MMPr on viral loads in the periphery and CNS. Pre-treatment with 6MMPr had no significant effect on viremia or viral titers in the periphery, but resulted in significantly higher viral loads in the brain, suggesting that the effect of 6MMPr is tissue-dependent. In conclusion, despite being a potent inhibitor of flaviviruses in cell culture, 6MMPr was not effective against West Nile disease in mice; however, further studies are warranted to reduce the toxicity and/or improve the bioavailability of this potential antiviral drug. PMID:22039536

  5. Inapparent Viral Infection of Cells In Vitro III. Manifestations of Infection of L Mouse Cells by Japanese Encephalitis Virus1

    PubMed Central

    Dubbs, D. R.; Scherer, W. F.

    1966-01-01

    Dubbs, D. R. (University of Minnesota, Minneapolis), and W. F. Scherer. Inapparent viral infection of cells in vitro. III. Manifestations of infection of L mouse cells by Japanese encephalitis virus. J. Bacteriol. 91:2349–2355. 1966.—Nine strains of Japanese encephalitis (JE) virus were propagated serially in cultures of L cells reaching titers of 103.5 to 106.3. Although cytopathic effects were not seen in cultures of contiguous L cells after infection with JE virus, cell growth was inhibited. Moreover, cell destruction was readily apparent in infected cultures of sparse, noncontiguous L cells. Differences in the size of cell population of infected and noninfected cultures (i) occurred despite only 0.2 to 3.5% of the cells in infected cultures being associated with infectious virus, (ii) were greater in actively growing cultures than in those kept in maintenance media, and (iii) were probably in part related to an interferon produced in infected cultures. Images PMID:4287589

  6. CD8+ T cells control Ross River virus infection in musculoskeletal tissues of infected mice.

    PubMed

    Burrack, Kristina S; Montgomery, Stephanie A; Homann, Dirk; Morrison, Thomas E

    2015-01-15

    Ross River virus (RRV), chikungunya virus, and related alphaviruses cause debilitating polyarthralgia and myalgia. Mouse models of RRV and chikungunya virus have demonstrated a role for the adaptive immune response in the control of these infections. However, questions remain regarding the role for T cells in viral control, including the magnitude, location, and dynamics of CD8(+) T cell responses. To address these questions, we generated a recombinant RRV expressing the H-2(b)-restricted glycoprotein 33 (gp33) determinant derived from the glycoprotein of lymphocytic choriomeningitis virus. Using tetramers, we tracked gp33-specific CD8(+) T cells during RRV-lymphocytic choriomeningitis virus infection. We found that acute RRV infection induces activation of CD8(+) T cell responses in lymphoid and musculoskeletal tissues that peak from 10-14 d postinoculation, suggesting that CD8(+) T cells contribute to control of acute RRV infection. Mice genetically deficient for CD8(+) T cells or wild-type mice depleted of CD8(+) T cells had elevated RRV loads in skeletal muscle tissue, but not joint-associated tissues, at 14 d postinoculation, suggesting that the ability of CD8(+) T cells to control RRV infection is tissue dependent. Finally, adoptively transferred T cells were capable of reducing RRV loads in skeletal muscle tissue of Rag1(-/-) mice, indicating that T cells can contribute to the control of RRV infection in the absence of B cells and Ab. Collectively, these data demonstrate a role for T cells in the control of RRV infection and suggest that the antiviral capacity of T cells is controlled in a tissue-specific manner. PMID:25488988

  7. Characterization of dengue virus 2 growth in megakaryocyte-erythrocyte progenitor cells.

    PubMed

    Clark, Kristina B; Hsiao, Hui-Mien; Bassit, Leda; Crowe, James E; Schinazi, Raymond F; Perng, Guey Chuen; Villinger, Francois

    2016-06-01

    Megakaryocyte-erythrocyte progenitor (MEP) cells are potential in vivo targets of dengue virus (DENV); the virus has been found associated with megakaryocytes ex vivo and platelets during DENV-induced thrombocytopenia. We report here that DENV serotype 2 (DENV2) propagates well in human nondifferentiated MEP cell lines (Meg01 and K562). In comparison to virus propagated in Vero cells, viruses from MEP cell lines had similar structure and buoyant density. However, differences in MEP-DENV2 stability and composition were suggested by distinct protein patterns in western blot analysis. Also, antibody neutralization of envelope domain I/II on MEP-DENV2 was reduced relative to that on Vero-DENV2. Infectious DENV2 was produced at comparable kinetics and magnitude in MEP and Vero cells. However, fewer virion structures appeared in electron micrographs of MEP cells. We propose that DENV2 infects and produces virus efficiently in megakaryocytes and that megakaryocyte impairment might contribute to dengue disease pathogenesis. PMID:27058763

  8. The Influence of Virus Infection on the Extracellular pH of the Host Cell Detected on Cell Membrane

    PubMed Central

    Liu, Hengjun; Maruyama, Hisataka; Masuda, Taisuke; Honda, Ayae; Arai, Fumihito

    2016-01-01

    Influenza virus infection can result in changes in the cellular ion levels at 2–3 h post-infection. More H+ is produced by glycolysis, and the viral M2 proton channel also plays a role in the capture and release of H+ during both viral entry and egress. Then the cells might regulate the intracellular pH by increasing the export of H+ from the intracellular compartment. Increased H+ export could lead indirectly to increased extracellular acidity. To detect changes in extracellular pH of both virus-infected and uninfected cells, pH sensors were synthesized using polystyrene beads (ϕ1 μm) containing Rhodamine B and Fluorescein isothiocyanate (FITC). The fluorescence intensity of FITC can respond to both pH