Coronavirus infections in horses in Saudi Arabia and Oman.

Science.gov (United States)

Hemida, M G; Chu, D K W; Perera, R A P M; Ko, R L W; So, R T Y; Ng, B C Y; Chan, S M S; Chu, S; Alnaeem, A A; Alhammadi, M A; Webby, R J; Poon, L L M; Balasuriya, U B R; Peiris, M

2017-12-01

Equine coronaviruses (ECoV) are the only coronavirus known to infect horses. So far, data on ECoV infection in horses remain limited to the USA, France and Japan and its geographic distribution is not well understood. We carried out RT-PCR on 306 nasal and 315 rectal swabs and tested 243 sera for antibodies to detect coronavirus infections in apparently healthy horses in Saudi Arabia and Oman. We document evidence of infection with ECoV and HKU23 coronavirus by RT-PCR. There was no conclusive evidence of Middle East respiratory syndrome coronavirus infection in horses. Serological data suggest that lineage A betacoronavirus infections are commonly infecting horses in Saudi Arabia and Oman but antibody cross-reactivities between these viruses do not permit us to use serological data alone to identify which coronaviruses are causing these infections. © 2017 Blackwell Verlag GmbH.

Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

Science.gov (United States)

Lee, Saemi; Jo, Seong-Deok; Son, Kidong; An, Injung; Jeong, Jipseol; Wang, Seung-Jun; Kim, Yongkwan; Jheong, Weonhwa; Oem, Jae-Ku

2018-01-01

Bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (SARS), coronavirus, and other coronaviruses found in mammals. However, little research has been conducted on bat coronaviruses in South Korea. In this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in South Korea. RT-PCR and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. Coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family Rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family Vespertiliodae. To determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the RNA-dependent RNA polymerase (RdRp) gene were analyzed phylogenetically. Thirteen sequences belonging to SARS-like betacoronaviruses showed the highest nucleotide identity (97.1-99.7%) with Bat-CoV-JTMC15 reported in China. The other five sequences were most similar to MERS-like betacoronaviruses. Four nucleotide sequences displayed the highest identity (94.1-95.1%) with Bat-CoV-HKU5 from Hong Kong. The one sequence from a carcass showed the highest nucleotide identity (99%) with Bat-CoV-SC2013 from China. These results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs.

Coronavirus 229E-related pneumonia in immunocompromised patients.

Science.gov (United States)

Pene, Frédéric; Merlat, Annabelle; Vabret, Astrid; Rozenberg, Flore; Buzyn, Agnès; Dreyfus, François; Cariou, Alain; Freymuth, François; Lebon, Pierre

2003-10-01

Coronaviruses strains 229E and OC43 have been associated with various respiratory illnesses ranging from the self-resolving common cold to severe pneumonia. Although chronic underlying conditions are major determinants of severe respiratory virus infections, few data about coronavirus-related pneumonia in immunocompromised patients are available. Here we report 2 well-documented cases of pneumonia related to coronavirus 229E, each with a different clinical presentation. Diagnosis was made on the basis of viral culture and electron microscopy findings that exhibited typical crown-like particles and through amplification of the viral genome by reverse transcriptase-polymerase chain reaction. On the basis of this report, coronaviruses should be considered as potential causative microorganisms of pneumonia in immunocompromised patients.

Transmission of Middle East respiratory syndrome coronavirus ...

African Journals Online (AJOL)

... hand hygiene, and cough etiquette, would minimize the infection rate among HCPs. The required consumables for maintaining hand hygiene should be readily available to all HCPs. Keywords: Middle East respiratory syndrome coronavirus (MERS-CoV), Systematic review, healthcareassociated infections, Coronaviruses ...

BIOLOGICAL CLONING OF A BOVINE CORONAVIRUS ISOLATE

OpenAIRE

Betancourt, A; Rodríguez, Edisleidy; Relova, Damarys; Barrera, Maritza

2008-01-01

Con el objetivo de obtener un aislado de Coronavirus bovino clonado biológicamente se adaptó el aislado VB73/04 a la multiplicación en la línea celular MDBK. Este aislado indujo la formación de placas, las cuales resultaron homogéneas después del clonaje biológico. La población viral obtenida fue identificada como Coronavirus bovino por RT-PCR y Seroneutralización. In order to obtain a biologically cloned bovine coronavirus isolate, the isolate VB73/04 was adapted to multiplication in MDBK...

Coronavirus Infection and Diversity in Bats in the Australasian Region.

Science.gov (United States)

Smith, C S; de Jong, C E; Meers, J; Henning, J; Wang, L- F; Field, H E

2016-03-01

Following the SARS outbreak, extensive surveillance was undertaken globally to detect and identify coronavirus diversity in bats. This study sought to identify the diversity and prevalence of coronaviruses in bats in the Australasian region. We identified four different genotypes of coronavirus, three of which (an alphacoronavirus and two betacoronaviruses) are potentially new species, having less than 90% nucleotide sequence identity with the most closely related described viruses. We did not detect any SARS-like betacoronaviruses, despite targeting rhinolophid bats, the putative natural host taxa. Our findings support the virus-host co-evolution hypothesis, with the detection of Miniopterus bat coronavirus HKU8 (previously reported in Miniopterus species in China, Hong Kong and Bulgaria) in Australian Miniopterus species. Similarly, we detected a novel betacoronavirus genotype from Pteropus alecto which is most closely related to Bat coronavirus HKU9 identified in other pteropodid bats in China, Kenya and the Philippines. We also detected possible cross-species transmission of bat coronaviruses, and the apparent enteric tropism of these viruses. Thus, our findings are consistent with a scenario wherein the current diversity and host specificity of coronaviruses reflects co-evolution with the occasional host shift.

A molecular arms race between host innate antiviral response and emerging human coronaviruses.

Science.gov (United States)

Wong, Lok-Yin Roy; Lui, Pak-Yin; Jin, Dong-Yan

2016-02-01

Coronaviruses have been closely related with mankind for thousands of years. Community-acquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

NARCIS (Netherlands)

ter Meulen, Jan; Bakker, Alexander B. H.; van den Brink, Edward N.; Weverling, Gerrit J.; Martina, Byron E. E.; Haagmans, Bart L.; Kuiken, Thijs; de Kruif, John; Preiser, Wolfgang; Spaan, Willy; Gelderblom, Hans R.; Goudsmit, Jaap; Osterhaus, Albert D. M. E.

2004-01-01

SARS coronavirus continues to cause sporadic cases of severe acute respiratory syndrome (SARS) in China. No active or passive immunoprophylaxis for disease induced by SARS coronavirus is available. We investigated prophylaxis of SARS coronavirus infection with a neutralising human monoclonal

Coronavirus minus-strand RNA synthesis and effect of cycloheximide on coronavirus RNA synthesis

International Nuclear Information System (INIS)

Sawicki, S.G.; Sawicki, D.L.

1986-01-01

The temporal sequence of coronavirus plus-strand and minus-strand RNA synthesis was determined in 17CL1 cells infected with the A59 strain of mouse hepatitis virus (MHV). MHV-induced fusion was prevented by keeping the pH of the medium below pH 6.8. This had no effect on the MHV replication cycle, but gave 5- to 10-fold-greater titers of infectious virus and delayed the detachment of cells from the monolayer which permitted viral RNA synthesis to be studied conveniently until at least 10 h postinfection. Seven species of poly(A)-containing viral RNAs were synthesized at early and late times infection, in nonequal but constant ratios. MHV minus-strand RNA synthesis was first detected at about 3 h after infection and was found exclusively in the viral replicative intermediates and was not detected in 60S single-stranded form in infected cells. Early in the replication cycle, from 45 to 65% of the [ 3 H]uridine pulse-labeled RF core of purified MHV replicative intermediates was in minus-strand RNA. The rate of minus-strand synthesis peaked at 5 to 6 h postinfection and then declined to about 20% of the maximum rate. The addition of cycloheximide before 3 h postinfection prevented viral RNA synthesis, whereas the addition of cycloheximide after viral RNA synthesis had begun resulted in the inhibition of viral RNA synthesis. The synthesis of both genome and subgenomic mRNAs and of viral minus strands required continued protein synthesis, and minis-strand RNA synthesis was three- to fourfold more sensitive to inhibition of cycloheximide than was plus-strand synthesis

Recombination in Avian Gamma-Coronavirus Infectious Bronchitis Virus

Directory of Open Access Journals (Sweden)

Mark W. Jackwood

2011-09-01

Full Text Available Recombination in the family Coronaviridae has been well documented and is thought to be a contributing factor in the emergence and evolution of different coronaviral genotypes as well as different species of coronavirus. However, there are limited data available on the frequency and extent of recombination in coronaviruses in nature and particularly for the avian gamma-coronaviruses where only recently the emergence of a turkey coronavirus has been attributed solely to recombination. In this study, the full-length genomes of eight avian gamma-coronavirus infectious bronchitis virus (IBV isolates were sequenced and along with other full-length IBV genomes available from GenBank were analyzed for recombination. Evidence of recombination was found in every sequence analyzed and was distributed throughout the entire genome. Areas that have the highest occurrence of recombination are located in regions of the genome that code for nonstructural proteins 2, 3 and 16, and the structural spike glycoprotein. The extent of the recombination observed, suggests that this may be one of the principal mechanisms for generating genetic and antigenic diversity within IBV. These data indicate that reticulate evolutionary change due to recombination in IBV, likely plays a major role in the origin and adaptation of the virus leading to new genetic types and strains of the virus.

Spike Protein Fusion Peptide and Feline Coronavirus Virulence

Science.gov (United States)

Chang, Hui-Wen; Egberink, Herman F.; Halpin, Rebecca; Spiro, David J.

2012-01-01

Coronaviruses are well known for their potential to change their host or tissue tropism, resulting in unpredictable new diseases and changes in pathogenicity; severe acute respiratory syndrome and feline coronaviruses, respectively, are the most recognized examples. Feline coronaviruses occur as 2 pathotypes: nonvirulent feline enteric coronaviruses (FECVs), which replicate in intestinal epithelium cells, and lethal feline infectious peritonitis viruses (FIPVs), which replicate in macrophages. Evidence indicates that FIPV originates from FECV by mutation, but consistent distinguishing differences have not been established. We sequenced the full genome of 11 viruses of each pathotype and then focused on the single most distinctive site by additionally sequencing hundreds of viruses in that region. As a result, we identified 2 alternative amino acid differences in the putative fusion peptide of the spike protein that together distinguish FIPV from FECV in >95% of cases. By these and perhaps other mutations, the virus apparently acquires its macrophage tropism and spreads systemically. PMID:22709821

Unraveling the Mysteries of Middle East Respiratory Syndrome Coronavirus

Centers for Disease Control (CDC) Podcasts

2014-03-11

Dr. Aron Hall, a CDC coronavirus epidemiologist, discusses Middle East Respiratory Syndrome Coronavirus.  Created: 3/11/2014 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 3/11/2014.

Coronavirus infection in mink (Mustela vison). Serological evidence of infection with a coronavirus related to transmissible gastroenteritis virus and porcine epidemic diarrhea virus

DEFF Research Database (Denmark)

Have, P; Moving, V; Svansson, V

1992-01-01

Antibodies to a transmissible gastroenteritis virus (TGEV)-related coronavirus have been demonstrated in mink sera by indirect immunofluorescence, peroxidase-linked antibody assays and immunoblotting. This is the first serological evidence of a specific coronavirus infection in mink. The putative...

Comparative properties of feline coronaviruses in vitro.

OpenAIRE

McKeirnan, A J; Evermann, J F; Davis, E V; Ott, R L

1987-01-01

Two feline coronaviruses were characterized to determine their biological properties in vitro and their antigenic relatedness to a previously recognized feline infectious peritonitis virus and canine coronavirus. The viruses, designated WSU 79-1146 and WSU 79-1683, were shown to have comparable growth curves with the prototype feline infectious peritonitis virus. Treatment of the feline infectious peritonitis virus strains with 0.25% trypsin indicated that they were relatively resistant to pr...

Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

Energy Technology Data Exchange (ETDEWEB)

Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

2011-09-28

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

Suppression of Coronavirus Replication by Cyclophilin Inhibitors

Directory of Open Access Journals (Sweden)

Takashi Sasaki

2013-05-01

Full Text Available Coronaviruses infect a variety of mammalian and avian species and cause serious diseases in humans, cats, mice, and birds in the form of severe acute respiratory syndrome (SARS, feline infectious peritonitis (FIP, mouse hepatitis, and avian infectious bronchitis, respectively. No effective vaccine or treatment has been developed for SARS-coronavirus or FIP virus, both of which cause lethal diseases. It has been reported that a cyclophilin inhibitor, cyclosporin A (CsA, could inhibit the replication of coronaviruses. CsA is a well-known immunosuppressive drug that binds to cellular cyclophilins to inhibit calcineurin, a calcium-calmodulin-activated serine/threonine-specific phosphatase. The inhibition of calcineurin blocks the translocation of nuclear factor of activated T cells from the cytosol into the nucleus, thus preventing the transcription of genes encoding cytokines such as interleukin-2. Cyclophilins are peptidyl-prolyl isomerases with physiological functions that have been described for many years to include chaperone and foldase activities. Also, many viruses require cyclophilins for replication; these include human immunodeficiency virus, vesicular stomatitis virus, and hepatitis C virus. However, the molecular mechanisms leading to the suppression of viral replication differ for different viruses. This review describes the suppressive effects of CsA on coronavirus replication.

Antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine.

Science.gov (United States)

Nemoto, Manabu; Kanno, Toru; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kokado, Hiroshi

2017-11-17

A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future.

Genetic diversity of coronaviruses in bats in Lao PDR and Cambodia.

Science.gov (United States)

Lacroix, Audrey; Duong, Veasna; Hul, Vibol; San, Sorn; Davun, Hull; Omaliss, Keo; Chea, Sokha; Hassanin, Alexandre; Theppangna, Watthana; Silithammavong, Soubanh; Khammavong, Kongsy; Singhalath, Sinpakone; Greatorex, Zoe; Fine, Amanda E; Goldstein, Tracey; Olson, Sarah; Joly, Damien O; Keatts, Lucy; Dussart, Philippe; Afelt, Aneta; Frutos, Roger; Buchy, Philippe

2017-03-01

South-East Asia is a hot spot for emerging zoonotic diseases, and bats have been recognized as hosts for a large number of zoonotic viruses such as Severe Acute Respiratory Syndrome (SARS), responsible for acute respiratory syndrome outbreaks. Thus, it is important to expand our knowledge of the presence of viruses in bats which could represent a risk to humans. Coronaviruses (CoVs) have been reported in bat species from Thailand, China, Indonesia, Taiwan and the Philippines. However no such work was conducted in Cambodia or Lao PDR. Between 2010 and 2013, 1965 bats were therefore sampled at interfaces with human populations in these two countries. They were tested for the presence of coronavirus by consensus reverse transcription-PCR assay. A total of 93 samples (4.7%) from 17 genera of bats tested positive. Sequence analysis revealed the presence of potentially 37 and 56 coronavirus belonging to alpha-coronavirus (αCoV) and beta-CoV (βCoV), respectively. The βCoVs group is known to include some coronaviruses highly pathogenic to human, such as SARS-CoV and MERS-CoV. All coronavirus sequences generated from frugivorous bats (family Pteropodidae) (n=55) clustered with other bat βCoVs of lineage D, whereas one coronavirus from Pipistrellus coromandra fell in the lineage C of βCoVs which also includes the MERS-CoV. αCoVs were all detected in various genera of insectivorous bats and clustered with diverse bat αCoV sequences previously published. A closely related strain of PEDV, responsible for severe diarrhea in pigs (PEDV-CoV), was detected in 2 Myotis bats. We highlighted the presence and the high diversity of coronaviruses circulating in bats from Cambodia and Lao PDR. Three new bat genera and species were newly identified as host of coronaviruses, namely Macroglossus sp., Megaerops niphanae and Myotis horsfieldii. Copyright © 2016 Elsevier B.V. All rights reserved.

Mechanisms of Coronavirus Cell Entry Mediated by the Viral Spike Protein

Directory of Open Access Journals (Sweden)

Gary R. Whittaker

2012-06-01

Full Text Available Coronaviruses are enveloped positive-stranded RNA viruses that replicate in the cytoplasm. To deliver their nucleocapsid into the host cell, they rely on the fusion of their envelope with the host cell membrane. The spike glycoprotein (S mediates virus entry and is a primary determinant of cell tropism and pathogenesis. It is classified as a class I fusion protein, and is responsible for binding to the receptor on the host cell as well as mediating the fusion of host and viral membranes—A process driven by major conformational changes of the S protein. This review discusses coronavirus entry mechanisms focusing on the different triggers used by coronaviruses to initiate the conformational change of the S protein: receptor binding, low pH exposure and proteolytic activation. We also highlight commonalities between coronavirus S proteins and other class I viral fusion proteins, as well as distinctive features that confer distinct tropism, pathogenicity and host interspecies transmission characteristics to coronaviruses.

Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

Directory of Open Access Journals (Sweden)

Yutaka Terada

Full Text Available Type II feline coronavirus (FCoV emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV. In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3'-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5'-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-Reactivity with SARS Coronavirus

Directory of Open Access Journals (Sweden)

David M Patrick

2006-01-01

Full Text Available BACKGROUND: In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV.

[Nosocomial infections due to human coronaviruses in the newborn].

Science.gov (United States)

Gagneur, A; Legrand, M C; Picard, B; Baron, R; Talbot, P J; de Parscau, L; Sizun, J

2002-01-01

Human coronaviruses, with two known serogroups named 229-E and OC-43, are enveloped positive-stranded RNA viruses. The large RNA is surrounded by a nucleoprotein (protein N). The envelop contains 2 or 3 glycoproteins: spike protein (or protein S), matrix protein (or protein M) and a hemagglutinin (or protein HE). Their pathogen role remains unclear because their isolation is difficult. Reliable and rapid methods as immunofluorescence with monoclonal antibodies and reverse transcription-polymerase chain reaction allow new researches on epidemiology. Human coronaviruses can survive for as long as 6 days in suspension and 3 hours after drying on surfaces, suggesting that they could be a source of hospital-acquired infections. Two prospective studies conducted in a neonatal and paediatric intensive care unit demonstrated a significant association of coronavirus-positive nasopharyngal samples with respiratory illness in hospitalised preterm neonates. Positive samples from staff suggested either a patient-to-staff or a staff-to-patient transmission. No cross-infection were observed from community-acquired respiratory-syncitial virus or influenza-infected children to neonates. Universal precautions with hand washing and surface desinfection could be proposed to prevent coronavirus transmission.

Detection of a group 2 coronavirus in dogs with canine infectious respiratory disease

International Nuclear Information System (INIS)

Erles, Kerstin; Toomey, Crista; Brooks, Harriet W.; Brownlie, Joe

2003-01-01

An investigation into the causes of canine infectious respiratory disease was carried out in a large rehoming kennel. Tissue samples taken from the respiratory tract of diseased dogs were tested for the presence of coronaviruses using RT-PCR with conserved primers for the polymerase gene. Sequence analysis of four positive samples showed the presence of a coronavirus with high similarity to both bovine and human coronavirus (strain OC43) in their polymerase and spike genes, whereas there was a low similarity to comparable genes in the enteric canine coronavirus. This canine respiratory coronavirus (CRCV) was detected by RT-PCR in 32/119 tracheal and 20/119 lung samples, with the highest prevalence being detected in dogs with mild clinical symptoms. Serological analysis showed that the presence of antibodies against CRCV on the day of entry into the kennel decreased the risk of developing respiratory disease

The coronavirus spike protein : mechanisms of membrane fusion and virion incorporation

NARCIS (Netherlands)

Bosch, B.J.

2004-01-01

The coronavirus spike protein is a membrane-anchored glycoprotein responsible for virus-cell attachment and membrane fusion, prerequisites for a successful virus infection. In this thesis, two aspects are described regarding the molecular biology of the coronavirus spike protein: its membrane fusion

Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

Directory of Open Access Journals (Sweden)

Blom Nikolaj

2004-06-01

Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

On the biased nucleotide composition of the human coronavirus RNA genome

NARCIS (Netherlands)

Berkhout, Ben; van Hemert, Formijn

2015-01-01

We investigated the nucleotide composition of the RNA genome of the six human coronaviruses. Some general coronavirus characteristics were apparent (e.g. high U, low C count), but we also detected species-specific signatures. Most strikingly, the high U and low C proportions are quite variable and

Coronaviruses in brain tissue from patients with multiple sclerosis

DEFF Research Database (Denmark)

Dessau, R B; Lisby, G; Frederiksen, J L

2001-01-01

Brain tissue from 25 patients with clinically definite multiple sclerosis (MS) and as controls brain tissue from 36 patients without neurological disease was tested for the presence of human coronaviral RNA. Four PCR assays with primers specific for N-protein of human coronavirus strain 229E...... and three PCR assays with primers specific for the nucleocapsid protein of human coronavirus strain OC43 were performed. Sporadic positive PCR assays were observed in both patients and controls in some of the PCR assays. However, these results were not reproducible and there was no difference...... in the proportion of positive signals from the MS patients compared to controls. Evidence for a chronic infection with the human coronaviruses strain 229E or OC43 in brain tissue from patients with MS or controls has not been found in this study....

Identification of Alpha and Beta Coronavirus in Wildlife Species in France: Bats, Rodents, Rabbits, and Hedgehogs

Directory of Open Access Journals (Sweden)

Elodie Monchatre-Leroy

2017-11-01

Full Text Available Coronaviruses are closely monitored in the context of emerging diseases and, as illustrated with Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and Middle East Respiratory Syndrome-coronavirus (MERS-CoV, are known to cross the species barrier and eventually to move from wildlife to humans. Knowledge of the diversity of coronaviruses in wildlife is therefore essential to better understand and prevent emergence events. This study explored the presence of coronaviruses in four wild mammal orders in France: Bats, rodents, lagomorphs, and hedgehogs. Betacoronavirus and Alphacoronavirus genera were identified. The results obtained suggest the circulation of potentially evolving virus strains, with the potential to cross the species barrier.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Directory of Open Access Journals (Sweden)

Eveline Kindler

2017-02-01

Full Text Available Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I. This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU activity is key to prevent early induction of double-stranded RNA (dsRNA host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

The SARS-unique domain (SUD of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

Directory of Open Access Journals (Sweden)

Jinzhi Tan

2009-05-01

Full Text Available Since the outbreak of severe acute respiratory syndrome (SARS in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV, the non-structural proteins (Nsps, have been determined. However, within the large Nsp3 (1922 amino-acid residues, the structure and function of the so-called SARS-unique domain (SUD have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core" of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively revealed that SUD(core forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins

Human coronavirus NL63, France

NARCIS (Netherlands)

Vabret, Astrid; Mourez, Thomas; Dina, Julia; van der Hoek, Lia; Gouarin, Stéphanie; Petitjean, Joëlle; Brouard, Jacques; Freymuth, François

2005-01-01

The human coronavirus NL63 (HCoV-NL63) was first identified in The Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory

Evidence for an ancestral association of human coronavirus 229E with bats

Czech Academy of Sciences Publication Activity Database

Corman, V. M.; Baldwin, H. J.; Tateno, A. F.; Zerbinati, R. M.; Annan, A.; Owusu, M.; Nkrumah, E. E.; Maganga, G. D.; Oppong, S.; Adu-Sarkodie, Y.; Vallo, Peter; da Silva Filho, L. V. R. F.; Leroy, E. M.; Thiel, V.; van der Hoek, L.; Poon, L. L. M.; Tschapka, M.; Drosten, C.; Drexler, J. F.

2015-01-01

Roč. 89, č. 23 (2015), s. 11858-11870 ISSN 0022-538X Institutional support: RVO:68081766 Keywords : respiratory syndrome coronavirus * SARS-coronavirus * genomic characterization * dromedary camels * clinical impact * virus * children * protein * spike * classification Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 4.606, year: 2015

Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

Energy Technology Data Exchange (ETDEWEB)

Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

2010-03-04

NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

Genomic Analysis and Surveillance of the Coronavirus Dominant in Ducks in China.

Directory of Open Access Journals (Sweden)

Qing-Ye Zhuang

Full Text Available The genetic diversity, evolution, distribution, and taxonomy of some coronaviruses dominant in birds other than chickens remain enigmatic. In this study we sequenced the genome of a newly identified coronavirus dominant in ducks (DdCoV, and performed a large-scale surveillance of coronaviruses in chickens and ducks using a conserved RT-PCR assay. The viral genome harbors a tandem repeat which is rare in vertebrate RNA viruses. The repeat is homologous to some proteins of various cellular organisms, but its origin remains unknown. Many substitutions, insertions, deletions, and some frameshifts and recombination events have occurred in the genome of the DdCoV, as compared with the coronavirus dominant in chickens (CdCoV. The distances between DdCoV and CdCoV are large enough to separate them into different species within the genus Gammacoronavirus. Our surveillance demonstrated that DdCoVs and CdCoVs belong to different lineages and occupy different ecological niches, further supporting that they should be classified into different species. Our surveillance also demonstrated that DdCoVs and CdCoVs are prevalent in live poultry markets in some regions of China. In conclusion, this study shed novel insight into the genetic diversity, evolution, distribution, and taxonomy of the coronaviruses circulating in chickens and ducks.

Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands (Coronavirus-like particles in rabbits).

NARCIS (Netherlands)

A.D.M.E. Osterhaus (Albert); J.S. Teppema; G. van Steenis (Bert)

1982-01-01

textabstractVirus-like particles were identified from the plasma of rabbits which developed pleural effusion disease after inoculation with different strains of Treponema pallidum. These particles were considered coronavirus-like on the basis of their size, morphology, and buoyant density. Clinical

Human Coronaviruses 229E and NL63: Close Yet Still So Far

NARCIS (Netherlands)

Dijkman, Ronald; van der Hoek, Lia

2009-01-01

HCoV-NL63 and HCoV-229E are two of the four human coronaviruses that circulate worldwide. These two viruses are unique in their relationship towards each other. Phylogenetically, the viruses are more closely related to each other than to any other human coronavirus, yet they only share 65% sequence

MERS-coronavirus: From discovery to intervention

NARCIS (Netherlands)

W. Widagdo; N.M.A. Okba (Nisreen); V. Stalin Raj; B.L. Haagmans (Bart)

2017-01-01

textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) still causes outbreaks despite public awareness and implementation of health care measures, such as rapid viral diagnosis and patient quarantine. Here we describe the current epidemiological picture of MERS-CoV, focusing on humans

Canine coronaviruses: Epidemiology, evolution and pathobiology

NARCIS (Netherlands)

Decaro, N.

2009-01-01

Coronaviruses (CoVs; order Nidovirales, family Coronaviridae) are viruses exceptionally prone to genetic evolution through the continual accumulation of mutations and by homologous recombination between related members. CoVs are organised into three antigenic groups of which group 1 is subdivided in

Prevalence of Korean cats with natural feline coronavirus infections

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Lee Myoung-Heon

2011-09-01

Full Text Available Abstract Background Feline coronavirus is comprised of two pathogenic biotypes consisting of feline infectious peritonitis virus (FIPV and feline enteric coronavirus (FECV, which are both divided into two serotypes. To examine the prevalence of Korean cats infected with feline coronavirus (FCoV type I and II, fecal samples were obtained from 212 cats (107 pet and 105 feral in 2009. Results Fourteen cats were FCoV-positive, including infections with type I FCoV (n = 8, type II FCoV (n = 4, and types I and II co-infection (n = 2. Low seroprevalences (13.7%, 29/212 of FCoV were identified in chronically ill cats (19.3%, 16/83 and healthy cats (10.1%, 13/129. Conclusions Although the prevalence of FCoV infection was not high in comparison to other countries, there was a higher prevalence of type I FCoV in Korean felines. The prevalence of FCoV antigen and antibody in Korean cats are expected to gradually increase due to the rising numbers of stray and companion cats.

Dynamics of the coronavirus replicative structures

NARCIS (Netherlands)

Hagemeijer, M.C.

2011-01-01

Coronaviruses (CoV) are positive-strand RNA (+RNA) viruses that are important infectious agents in both animals and man. Upon infection, CoVs generate large multicomponent protein complexes, consisting of 16 nonstructural proteins (nsp’s) and yet to be identified cellular proteins, dedicated to the

Structure of the C-terminal domain of nsp4 from feline coronavirus

International Nuclear Information System (INIS)

Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh; Snijder, Eric J.; Gorbalenya, Alexander E.; Berglind, Hanna; Nordlund, Pär; Coutard, Bruno; Tucker, Paul A.

2009-01-01

The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4 3 . The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions

Structure of the C-terminal domain of nsp4 from feline coronavirus

Energy Technology Data Exchange (ETDEWEB)

Manolaridis, Ioannis; Wojdyla, Justyna A.; Panjikar, Santosh [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany); Snijder, Eric J.; Gorbalenya, Alexander E. [Molecular Virology Laboratory, Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden (Netherlands); Berglind, Hanna; Nordlund, Pär [Division of Biophysics, Department of Medical Biochemistry and Biophysics, Scheeles väg 2, Karolinska Institute, SE-171 77 Stockholm (Sweden); Coutard, Bruno [Laboratoire Architecture et Fonction des Macromolécules Biologiques, UMR 6098, AFMB-CNRS-ESIL, Case 925, 163 Avenue de Luminy, 13288 Marseille (France); Tucker, Paul A., E-mail: tucker@embl-hamburg.de [EMBL Hamburg Outstation, c/o DESY, Notkestrasse 85, D-22603 Hamburg (Germany)

2009-08-01

The structure of the cytosolic C-terminal domain of nonstructural protein 4 from feline coronavirus has been determined and analyzed. Coronaviruses are a family of positive-stranded RNA viruses that includes important pathogens of humans and other animals. The large coronavirus genome (26–31 kb) encodes 15–16 nonstructural proteins (nsps) that are derived from two replicase polyproteins by autoproteolytic processing. The nsps assemble into the viral replication–transcription complex and nsp3, nsp4 and nsp6 are believed to anchor this enzyme complex to modified intracellular membranes. The largest part of the coronavirus nsp4 subunit is hydrophobic and is predicted to be embedded in the membranes. In this report, a conserved C-terminal domain (∼100 amino-acid residues) has been delineated that is predicted to face the cytoplasm and has been isolated as a soluble domain using library-based construct screening. A prototypical crystal structure at 2.8 Å resolution was obtained using nsp4 from feline coronavirus. Unmodified and SeMet-substituted proteins were crystallized under similar conditions, resulting in tetragonal crystals that belonged to space group P4{sub 3}. The phase problem was initially solved by single isomorphous replacement with anomalous scattering (SIRAS), followed by molecular replacement using a SIRAS-derived composite model. The structure consists of a single domain with a predominantly α-helical content displaying a unique fold that could be engaged in protein–protein interactions.

Reverse genetics of SARS-related coronavirus using vaccinia virus-based recombination.

Directory of Open Access Journals (Sweden)

Sjoerd H E van den Worm

Full Text Available Severe acute respiratory syndrome (SARS is a zoonotic disease caused by SARS-related coronavirus (SARS-CoV that emerged in 2002 to become a global health concern. Although the original outbreak was controlled by classical public health measures, there is a real risk that another SARS-CoV could re-emerge from its natural reservoir, either in its original form or as a more virulent or pathogenic strain; in which case, the virus would be difficult to control in the absence of any effective antiviral drugs or vaccines. Using the well-studied SARS-CoV isolate HKU-39849, we developed a vaccinia virus-based SARS-CoV reverse genetic system that is both robust and biosafe. The SARS-CoV genome was cloned in separate vaccinia virus vectors, (vSARS-CoV-5prime and vSARS-CoV-3prime as two cDNAs that were subsequently ligated to create a genome-length SARS-CoV cDNA template for in vitro transcription of SARS-CoV infectious RNA transcripts. Transfection of the RNA transcripts into permissive cells led to the recovery of infectious virus (recSARS-CoV. Characterization of the plaques produced by recSARS-CoV showed that they were similar in size to the parental SARS-CoV isolate HKU-39849 but smaller than the SARS-CoV isolate Frankfurt-1. Comparative analysis of replication kinetics showed that the kinetics of recSARS-CoV replication are similar to those of SARS-CoV Frankfurt-1, although the titers of virus released into the culture supernatant are approximately 10-fold less. The reverse genetic system was finally used to generate a recSARS-CoV reporter virus expressing Renilla luciferase in order to facilitate the analysis of SARS-CoV gene expression in human dendritic cells (hDCs. In parallel, a Renilla luciferase gene was also inserted into the genome of human coronavirus 229E (HCoV-229E. Using this approach, we demonstrate that, in contrast to HCoV-229E, SARS-CoV is not able to mediate efficient heterologous gene expression in hDCs.

Coronaviruses in guano from Pteropus medius bats in Peradeniya, Sri Lanka.

Science.gov (United States)

Kudagammana, H D W S; Thevanesam, V; Chu, D K W; Eriyagama, N B; Peiris, J S M; Noordeen, F

2018-03-02

Bats are a unique group of mammals well suited to be hosts for emerging viruses. With current rates of deforestation and urbanization, redistribution of bat habitats to urban and suburban areas may bring bats into closer contact with livestock and humans. Common flying fox, Pteropus medius (previously known as Pteropus giganteus), forms large communal roosts on treetops, often in close proximity to human habitation in Sri Lanka. This report describes the detection of coronavirus RNA in P. medius bat guano collected in Peradeniya, Sri Lanka. These viruses had >97% nucleotide identity with coronaviruses detected in Cynopterus sphinx, Scotophilus heathii and S. kuhlii bats in Thailand. Pteropus medius is widespread in Asia and appears to excrete group D coronaviruses, which are hitherto confined to bats; however, these findings may have public health implications in the future. © 2018 Blackwell Verlag GmbH.

Coronavirus infection of polarized epithelial cells

NARCIS (Netherlands)

Rossen, J W; Horzinek, M C; Rottier, P J

1995-01-01

Epithelial cells are the first host cells to be infected by incoming c oronaviruses. Recent observations in vitro show that coronaviruses are released from a specific side of these polarized cells, and this polarized release might be important for the spread of the infection in vivo. Mechanisms for

Renin-angiotensin system in human coronavirus pathogenesis

NARCIS (Netherlands)

Wevers, Brigitte A.; van der Hoek, Lia

2010-01-01

Although initially considered relatively harmless pathogens, human coronaviruses (HCoVs) are nowadays known to be associated with more severe clinical complications. Still, their precise pathogenic potential is largely unknown, particularly regarding the most recently identified species HCoV-NL63

Influenza polymerase encoding mRNAs utilize atypical mRNA nuclear export.

Science.gov (United States)

Larsen, Sean; Bui, Steven; Perez, Veronica; Mohammad, Adeba; Medina-Ramirez, Hilario; Newcomb, Laura L

2014-08-28

Influenza is a segmented negative strand RNA virus. Each RNA segment is encapsulated by influenza nucleoprotein and bound by the viral RNA dependent RNA polymerase (RdRP) to form viral ribonucleoproteins responsible for RNA synthesis in the nucleus of the host cell. Influenza transcription results in spliced mRNAs (M2 and NS2), intron-containing mRNAs (M1 and NS1), and intron-less mRNAs (HA, NA, NP, PB1, PB2, and PA), all of which undergo nuclear export into the cytoplasm for translation. Most cellular mRNA nuclear export is Nxf1-mediated, while select mRNAs utilize Crm1. Here we inhibited Nxf1 and Crm1 nuclear export prior to infection with influenza A/Udorn/307/1972(H3N2) virus and analyzed influenza intron-less mRNAs using cellular fractionation and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). We examined direct interaction between Nxf1 and influenza intron-less mRNAs using immuno purification of Nxf1 and RT-PCR of associated RNA. Inhibition of Nxf1 resulted in less influenza intron-less mRNA export into the cytoplasm for HA and NA influenza mRNAs in both human embryonic kidney cell line (293 T) and human lung adenocarcinoma epithelial cell line (A549). However, in 293 T cells no change was observed for mRNAs encoding the components of the viral ribonucleoproteins; NP, PA, PB1, and PB2, while in A549 cells, only PA, PB1, and PB2 mRNAs, encoding the RdRP, remained unaffected; NP mRNA was reduced in the cytoplasm. In A549 cells NP, NA, HA, mRNAs were found associated with Nxf1 but PA, PB1, and PB2 mRNAs were not. Crm1 inhibition also resulted in no significant difference in PA, PB1, and PB2 mRNA nuclear export. These results further confirm Nxf1-mediated nuclear export is functional during the influenza life cycle and hijacked for select influenza mRNA nuclear export. We reveal a cell type difference for Nxf1-mediated nuclear export of influenza NP mRNA, a reminder that cell type can influence molecular mechanisms. Importantly, we

Detection of Coronaviruses in Bats of Various Species in Italy

Directory of Open Access Journals (Sweden)

Maria B. Boniotti

2013-10-01

Full Text Available Bats are natural reservoirs for many mammalian coronaviruses, which have received renewed interest after the discovery of the severe acute respiratory syndrome (SARS and the Middle East respiratory syndrome (MERS CoV in humans. This study describes the identification and molecular characterization of alphacoronaviruses and betacoronaviruses in bats in Italy, from 2010 to 2012. Sixty-nine faecal samples and 126 carcasses were tested using pan-coronavirus RT-PCR. Coronavirus RNAs were detected in seven faecal samples and nine carcasses. A phylogenetic analysis of RNA-dependent RNA polymerase sequence fragments aided in identifying two alphacoronaviruses from Kuhl’s pipistrelle (Pipistrellus kuhlii, three clade 2b betacoronaviruses from lesser horseshoe bats (Rhinolophus hipposideros, and 10 clade 2c betacoronaviruses from Kuhl’s pipistrelle, common noctule (Nyctalus noctula, and Savi’s pipistrelle (Hypsugo savii. This study fills a substantive gap in the knowledge on bat-CoV ecology in Italy, and extends the current knowledge on clade 2c betacoronaviruses with new sequences obtained from bats that have not been previously described as hosts of these viruses.

Transmission of MERS-coronavirus in household contacts

NARCIS (Netherlands)

Drosten, Christian; Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Al-Masri, Malak; Hossain, Raheela; Madani, Hosam; Sieberg, Andrea; Bosch, Berend Jan|info:eu-repo/dai/nl/273306049; Lattwein, Erik; Alhakeem, Raafat F; Assiri, Abdullah M; Hajomar, Waleed; Albarrak, Ali M; Al-Tawfiq, Jaffar A; Zumla, Alimuddin I; Memish, Ziad A

2014-01-01

BACKGROUND: Strategies to contain the Middle East respiratory syndrome coronavirus (MERS-CoV) depend on knowledge of the rate of human-to-human transmission, including subclinical infections. A lack of serologic tools has hindered targeted studies of transmission. METHODS: We studied 26 index

Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

Science.gov (United States)

Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

2013-01-01

Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

Human Coronaviruses 229E and NL63: Close Yet Still So Far

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Ronald Dijkman

2009-04-01

Full Text Available HCoV-NL63 and HCoV-229E are two of the four human coronaviruses that circulate worldwide. These two viruses are unique in their relationship towards each other. Phylogenetically, the viruses are more closely related to each other than to any other human coronavirus, yet they only share 65% sequence identity. Moreover, the viruses use different receptors to enter their target cell. HCoV-NL63 is associated with croup in children, whereas all signs suggest that the virus probably causes the common cold in healthy adults. HCoV-229E is a proven common cold virus in healthy adults, so it is probable that both viruses induce comparable symptoms in adults, even though their mode of infection differs. Here, we present an overview of the current knowledge on both human coronaviruses, focusing on similarities and differences.

[Prevalence and clinical characteristics of coronavirus NL63 infection in children hospitalized for acute lower respiratory tract infections in Changsha].

Science.gov (United States)

Zhang, Fei; Zhang, Bing; Xie, Zhi-Ping; Gao, Han-Chun; Zhao, Xin; Zhong, Li-Li; Zhou, Qiong-Hua; Hou, Yun-De; Duan, Zhao-Jun

2012-04-01

The main objective of this study was to explore the prevalence and clinical characteristics of human coronavirus NL63 infection in hospitalized children with acute lower respiratory tract infection (ALRTI) in Changsha. Nasopharyngeal aspirates (NPA) samples were collected from 1185 hospitalized children with ALRTI at the People's Hospital of Hunan province, between September 2008 and October 2010. Reverse transcriptase polymerase chain reaction (RT-PCR) was employed to screen for coronavirus NL63, which is a 255 bp fragment of a part of N gene. All positive amplification products were confirmed by sequencing and compared with those in GenBank. The overall frequency of coronavirus NL63 infection was 0.8%, 6 (60%) out of the coronavirus NL63 positive patients were detected in summer, 2 in autumn, 1 in spring and winter, respectively. The patients were from 2 months to two and a half years old. The clinical diagnosis was bronchopneumonia (60%), bronchiolitis (30%), and acute laryngotracheal bronchitis (10%). Four of the 10 cases had critical illness, 4 cases had underlying diseases, and 7 cases had mixed infection with other viruses. The homogeneity of coronavirus NL63 with those published in the GenBank at nucleotide levels was 97%-100%. Coronavirus NL63 infection exists in hospitalized children with acute lower respiratory tract infection in Changsha. Coronavirus NL63 infections are common in children under 3 years of age. There is significant difference in the infection rate between the boys and the girls: the boys had higher rate than the girls. The peak of prevalence of the coronavirus NL63 was in summer. A single genetic lineage of coronavirus NL63 was revealed in human subjects in Changsha. Coronavirus NL63 may also be one of the lower respiratory pathogen in China.

Inactivation of surrogate coronaviruses on hard surfaces by health care germicides.

Science.gov (United States)

Hulkower, Rachel L; Casanova, Lisa M; Rutala, William A; Weber, David J; Sobsey, Mark D

2011-06-01

In the 2003 severe acute respiratory syndrome outbreak, finding viral nucleic acids on hospital surfaces suggested surfaces could play a role in spread in health care environments. Surface disinfection may interrupt transmission, but few data exist on the effectiveness of health care germicides against coronaviruses on surfaces. The efficacy of health care germicides against 2 surrogate coronaviruses, mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV), was tested using the quantitative carrier method on stainless steel surfaces. Germicides were o-phenylphenol/p-tertiary amylphenol) (a phenolic), 70% ethanol, 1:100 sodium hypochlorite, ortho-phthalaldehyde (OPA), instant hand sanitizer (62% ethanol), and hand sanitizing spray (71% ethanol). After 1-minute contact time, for TGEV, there was a log(10) reduction factor of 3.2 for 70% ethanol, 2.0 for phenolic, 2.3 for OPA, 0.35 for 1:100 hypochlorite, 4.0 for 62% ethanol, and 3.5 for 71% ethanol. For MHV, log(10) reduction factors were 3.9 for 70% ethanol, 1.3 for phenolic, 1.7 for OPA, 0.62 for 1:100 hypochlorite, 2.7 for 62% ethanol, and 2.0 for 71% ethanol. Only ethanol reduced infectivity of the 2 coronaviruses by >3-log(10) after 1 minute. Germicides must be chosen carefully to ensure they are effective against viruses such as severe acute respiratory syndrome coronavirus. Copyright © 2011 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.

Identification of Aminopeptidase N as a Cellular Receptor for Human Coronavirus-229E

Science.gov (United States)

1992-05-12

hemagglutinating encephalomyelitis virus (HEV), canine coronavirus (CCV), cat FIPV and feline enteric corona virus (FECV), human CVLPs, mouse...While the cat , dog and pig serve as natural hosts for the other coronavirus group 1 viruses , feline infectious peritonitis virus (FIPV), canine...3 2 . Virus Receptors ••••••••.••••••.....•................ 20 3. Viruses Which Cause Common Colds

Molecular epidemiology of bovine coronavirus on the basis of comparative analyses of the S gene

DEFF Research Database (Denmark)

Liu, Lihong; Hägglund, Sara; Hakhverdyan, Mikhayil

2006-01-01

Bovine coronavirus (BCoV), a group 2 member of the genus Coronavirus in the family Coronaviridae, is an important pathogen in cattle worldwide. It causes diarrhea in adult animals (winter dysentery), as well as enteric and respiratory diseases in calves. The annual occurrence of BCoV epidemics...

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules

International Nuclear Information System (INIS)

Wu, H.-Y.; Guy, James S.; Yoo, Dongwan; Vlasak, Reinhard; Urbach, Ena; Brian, David A.

2003-01-01

5' and 3' UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5' UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5' UTR and adjacent 289-nt sequences, and 3' UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3' UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5' and 3' replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members

IRAK2 directs stimulus-dependent nuclear export of inflammatory mRNAs.

Science.gov (United States)

Zhou, Hao; Bulek, Katarzyna; Li, Xiao; Herjan, Tomasz; Yu, Minjia; Qian, Wen; Wang, Han; Zhou, Gao; Chen, Xing; Yang, Hui; Hong, Lingzi; Zhao, Junjie; Qin, Luke; Fukuda, Koichi; Flotho, Annette; Gao, Ji; Dongre, Ashok; Carman, Julie A; Kang, Zizhen; Su, Bing; Kern, Timothy S; Smith, Jonathan D; Hamilton, Thomas A; Melchior, Frauke; Fox, Paul L; Li, Xiaoxia

2017-10-09

Expression of inflammatory genes is determined in part by post-transcriptional regulation of mRNA metabolism but how stimulus- and transcript-dependent nuclear export influence is poorly understood. Here, we report a novel pathway in which LPS/TLR4 engagement promotes nuclear localization of IRAK2 to facilitate nuclear export of a specific subset of inflammation-related mRNAs for translation in murine macrophages. IRAK2 kinase activity is required for LPS-induced RanBP2-mediated IRAK2 sumoylation and subsequent nuclear translocation. Array analysis showed that an SRSF1-binding motif is enriched in mRNAs dependent on IRAK2 for nuclear export. Nuclear IRAK2 phosphorylates SRSF1 to reduce its binding to target mRNAs, which promotes the RNA binding of the nuclear export adaptor ALYREF and nuclear export receptor Nxf1 loading for the export of the mRNAs. In summary, LPS activates a nuclear function of IRAK2 that facilitates the assembly of nuclear export machinery to export selected inflammatory mRNAs to the cytoplasm for translation.

Feline Coronavirus 3c Protein: A Candidate for a Virulence Marker?

Directory of Open Access Journals (Sweden)

A. S. Hora

2016-01-01

Full Text Available Feline infectious peritonitis virus (FIPV is highly virulent and responsible for the highly fatal disease feline infectious peritonitis (FIP, whereas feline enteric coronavirus (FECV is widespread among the feline population and typically causes asymptomatic infections. Some candidates for genetic markers capable of differentiating these two pathotypes of a unique virus (feline coronavirus have been proposed by several studies. In the present survey, in order to search for markers that can differentiate FECV and FIPV, several clones of the 3a–c, E, and M genes were sequenced from samples obtained from cats with or without FIP. All genes showed genetic diversity and suggested the presence of FCoV mutant spectrum capable of producing a virulent pathotype in an individual-specific way. In addition, all the feline coronavirus FIPV strains demonstrated a truncated 3c protein, and the 3c gene was the only observed pathotypic marker for FCoVs, showing that 3c gene is a candidate marker for the distinction between the two pathotypes when the mutant spectrum is taken into account.

Mosaic Evolution of the Severe Acute Respiratory Syndrome Coronavirus

Science.gov (United States)

Stavrinides, John; Guttman, David S.

2004-01-01

Severe acute respiratory syndrome (SARS) is a deadly form of pneumonia caused by a novel coronavirus, a viral family responsible for mild respiratory tract infections in a wide variety of animals including humans, pigs, cows, mice, cats, and birds. Analyses to date have been unable to identify the precise origin of the SARS coronavirus. We used Bayesian, neighbor-joining, and split decomposition phylogenetic techniques on the SARS virus replicase, surface spike, matrix, and nucleocapsid proteins to reveal the evolutionary origin of this recently emerging infectious agent. The analyses support a mammalian-like origin for the replicase protein, an avian-like origin for the matrix and nucleocapsid proteins, and a mammalian-avian mosaic origin for the host-determining spike protein. A bootscan recombination analysis of the spike gene revealed high nucleotide identity between the SARS virus and a feline infectious peritonitis virus throughout the gene, except for a 200- base-pair region of high identity to an avian sequence. These data support the phylogenetic analyses and suggest a possible past recombination event between mammalian-like and avian-like parent viruses. This event occurred near a region that has been implicated to be the human receptor binding site and may have been directly responsible for the switch of host of the SARS coronavirus from animals to humans. PMID:14671089

Discovery of a novel bottlenose dolphin coronavirus reveals a distinct species of marine mammal coronavirus in Gammacoronavirus.

Science.gov (United States)

Woo, Patrick C Y; Lau, Susanna K P; Lam, Carol S F; Tsang, Alan K L; Hui, Suk-Wai; Fan, Rachel Y Y; Martelli, Paolo; Yuen, Kwok-Yung

2014-01-01

While gammacoronaviruses mainly comprise infectious bronchitis virus (IBV) and its closely related bird coronaviruses (CoVs), the only mammalian gammacoronavirus was discovered from a white beluga whale (beluga whale CoV [BWCoV] SW1) in 2008. In this study, we discovered a novel gammacoronavirus from fecal samples from three Indo-Pacific bottlenose dolphins (Tursiops aduncus), which we named bottlenose dolphin CoV (BdCoV) HKU22. All the three BdCoV HKU22-positive samples were collected on the same date, suggesting a cluster of infection, with viral loads of 1 × 10(3) to 1 × 10(5) copies per ml. Clearance of virus was associated with a specific antibody response against the nucleocapsid of BdCoV HKU22. Complete genome sequencing and comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 have similar genome characteristics and structures. Their genome size is about 32,000 nucleotides, the largest among all CoVs, as a result of multiple unique open reading frames (NS5a, NS5b, NS5c, NS6, NS7, NS8, NS9, and NS10) between their membrane (M) and nucleocapsid (N) protein genes. Although comparative genome analysis showed that BdCoV HKU22 and BWCoV SW1 should belong to the same species, a major difference was observed in the proteins encoded by their spike (S) genes, which showed only 74.3 to 74.7% amino acid identities. The high ratios of the number of synonymous substitutions per synonymous site (Ks) to the number of nonsynonymous substitutions per nonsynonymous site (Ka) in multiple regions of the genome, especially the S gene (Ka/Ks ratio, 2.5), indicated that BdCoV HKU22 may be evolving rapidly, supporting a recent transmission event to the bottlenose dolphins. We propose a distinct species, Cetacean coronavirus, in Gammacoronavirus, to include BdCoV HKU22 and BWCoV SW1, whereas IBV and its closely related bird CoVs represent another species, Avian coronavirus, in Gammacoronavirus.

The SARS-coronavirus-host interactome: identification of cyclophilins as target for pan-coronavirus inhibitors.

Directory of Open Access Journals (Sweden)

Susanne Pfefferle

2011-10-01

Full Text Available Coronaviruses (CoVs are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS in 2002/2003 has demonstrated human vulnerability to (Coronavirus CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B as interaction partners of the CoV non-structural protein 1 (Nsp1. These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Cytoplasmic tail of coronavirus spike protein has intracellular

Indian Academy of Sciences (India)

https://www.ias.ac.in/article/fulltext/jbsc/042/02/0231-0244. Keywords. Coronavirus spike protein trafficking; cytoplasmic tail signal; endoplasmic reticulum–Golgi intermediate complex; lysosome. Abstract. Intracellular trafficking and localization studies of spike protein from SARS and OC43 showed that SARS spikeprotein is ...

Molecular and pathological identification of feline coronavirus type I

African Journals Online (AJOL)

DR. NJ TONUKARI

2012-06-05

Jun 5, 2012 ... In this study, we described the isolation and molecular characterization of .... fecv2b) designed in the regions of S-protein gene were used to differentiate ..... The molecular dynamics of feline coronaviruses. Vet. Microbiol.

Feline coronavirus replication is affected by both cyclophilin A and cyclophilin B.

Science.gov (United States)

Tanaka, Yoshikazu; Sato, Yuka; Sasaki, Takashi

2017-02-01

Feline coronavirus (FCoV) causes the fatal disease feline infectious peritonitis, which is currently incurable by drug treatment, and no effective vaccines are available. Cyclosporin A (CsA), a cyclophilin (Cyp) inhibitor, inhibits the replication of FCoV in vitro and in vivo as well as the replication of human and animal coronaviruses. However, the mechanism underlying the regulation of coronavirus replication by CsA is unknown. In this study, we analysed the role of Cyps in FCoV replication using knockdown and knockout cells specific to Cyps. Inhibition of CypA and CypB reduced FCoV replication, with replication in knockout cells being much less than that in knockdown cells. Furthermore, the proteins expressed by CypA and CypB harbouring mutations in their respective predicted peptidyl-prolyl cis-transisomerase active sites, which also alter the affinities between Cyps and CsA, inhibited FCoV replication. These findings indicate that the peptidyl-prolyl cis-transisomerase active sites of Cyps might be required for FCoV replication.

The emerging novel Middle East respiratory syndrome coronavirus: The “knowns” and “unknowns”

Directory of Open Access Journals (Sweden)

Jasper Fuk-Woo Chan

2013-07-01

Full Text Available A novel lineage C betacoronavirus, originally named human coronavirus EMC/2012 (HCoV-EMC and recently renamed Middle East respiratory syndrome coronavirus (MERS-CoV, that is phylogenetically closely related to Tylonycteris bat coronavirus HKU4 and Pipistrellus bat coronavirus HKU5, which we discovered in 2007 from bats in Hong Kong, has recently emerged in the Middle East to cause a severe acute respiratory syndrome (SARS-like infection in humans. The first laboratory-confirmed case, which involved a 60-year-old man from Bisha, the Kingdom of Saudi Arabia (KSA, who died of rapidly progressive community-acquired pneumonia and acute renal failure, was announced by the World Health Organization (WHO on September 23, 2012. Since then, a total of 70 cases, including 39 fatalities, have been reported in the Middle East and Europe. Recent clusters involving epidemiologically-linked household contacts and hospital contacts in the Middle East, Europe, and Africa strongly suggested possible human-to-human transmission. Clinical and laboratory research data generated in the past few months have provided new insights into the possible animal reservoirs, transmissibility, and virulence of MERS-CoV, and the optimal laboratory diagnostic options and potential antiviral targets for MERS-CoV-associated infection.

Development of Broad-Spectrum Halomethyl Ketone Inhibitors Against Coronavirus Main Protease 3CL(pro)

Energy Technology Data Exchange (ETDEWEB)

Bacha,U.; Barilla, J.; Gabelli, S.; Kiso, Y.; Amzel, L.; Freire, E.

2008-01-01

Coronaviruses comprise a large group of RNA viruses with diverse host specificity. The emergence of highly pathogenic strains like the SARS coronavirus (SARS-CoV), and the discovery of two new coronaviruses, NL-63 and HKU1, corroborates the high rate of mutation and recombination that have enabled them to cross species barriers and infect novel hosts. For that reason, the development of broad-spectrum antivirals that are effective against several members of this family is highly desirable. This goal can be accomplished by designing inhibitors against a target, such as the main protease 3CLpro (Mpro), which is highly conserved among all coronaviruses. Here 3CLpro derived from the SARS-CoV was used as the primary target to identify a new class of inhibitors containing a halomethyl ketone warhead. The compounds are highly potent against SARS 3CLpro with Ki's as low as 300 nm. The crystal structure of the complex of one of the compounds with 3CLpro indicates that this inhibitor forms a thioether linkage between the halomethyl carbon of the warhead and the catalytic Cys 145. Furthermore, Structure Activity Relationship (SAR) studies of these compounds have led to the identification of a pharmacophore that accurately defines the essential molecular features required for the high affinity.

Deficient incorporation of spike protein into virions contributes to the lack of infectivity following establishment of a persistent, non-productive infection in oligodendroglial cell culture by murine coronavirus

International Nuclear Information System (INIS)

Liu Yin; Herbst, Werner; Cao Jianzhong; Zhang Xuming

2011-01-01

Infection of mouse oligodendrocytes with a recombinant mouse hepatitis virus (MHV) expressing a green fluorescence protein facilitated specific selection of virus-infected cells and subsequent establishment of persistence. Interestingly, while viral genomic RNAs persisted in infected cells over 14 subsequent passages with concomitant synthesis of viral subgenomic mRNAs and structural proteins, no infectious virus was isolated beyond passage 2. Further biochemical and electron microscopic analyses revealed that virions, while assembled, contained little spike in the envelope, indicating that lack of infectivity during persistence was likely due to deficiency in spike incorporation. This type of non-lytic, non-productive persistence in oligodendrocytes is unique among animal viruses and resembles MHV persistence previously observed in the mouse central nervous system. Thus, establishment of such a culture system that can recapitulate the in vivo phenomenon will provide a powerful approach for elucidating the mechanisms of coronavirus persistence in glial cells at the cellular and molecular levels.

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.

Science.gov (United States)

Ge, Xing-Yi; Li, Jia-Lu; Yang, Xing-Lou; Chmura, Aleksei A; Zhu, Guangjian; Epstein, Jonathan H; Mazet, Jonna K; Hu, Ben; Zhang, Wei; Peng, Cheng; Zhang, Yu-Ji; Luo, Chu-Ming; Tan, Bing; Wang, Ning; Zhu, Yan; Crameri, Gary; Zhang, Shu-Yi; Wang, Lin-Fa; Daszak, Peter; Shi, Zheng-Li

2013-11-28

The 2002-3 pandemic caused by severe acute respiratory syndrome coronavirus (SARS-CoV) was one of the most significant public health events in recent history. An ongoing outbreak of Middle East respiratory syndrome coronavirus suggests that this group of viruses remains a key threat and that their distribution is wider than previously recognized. Although bats have been suggested to be the natural reservoirs of both viruses, attempts to isolate the progenitor virus of SARS-CoV from bats have been unsuccessful. Diverse SARS-like coronaviruses (SL-CoVs) have now been reported from bats in China, Europe and Africa, but none is considered a direct progenitor of SARS-CoV because of their phylogenetic disparity from this virus and the inability of their spike proteins to use the SARS-CoV cellular receptor molecule, the human angiotensin converting enzyme II (ACE2). Here we report whole-genome sequences of two novel bat coronaviruses from Chinese horseshoe bats (family: Rhinolophidae) in Yunnan, China: RsSHC014 and Rs3367. These viruses are far more closely related to SARS-CoV than any previously identified bat coronaviruses, particularly in the receptor binding domain of the spike protein. Most importantly, we report the first recorded isolation of a live SL-CoV (bat SL-CoV-WIV1) from bat faecal samples in Vero E6 cells, which has typical coronavirus morphology, 99.9% sequence identity to Rs3367 and uses ACE2 from humans, civets and Chinese horseshoe bats for cell entry. Preliminary in vitro testing indicates that WIV1 also has a broad species tropism. Our results provide the strongest evidence to date that Chinese horseshoe bats are natural reservoirs of SARS-CoV, and that intermediate hosts may not be necessary for direct human infection by some bat SL-CoVs. They also highlight the importance of pathogen-discovery programs targeting high-risk wildlife groups in emerging disease hotspots as a strategy for pandemic preparedness.

Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1.

Science.gov (United States)

Lu, Shuai; Wang, Yanqun; Chen, Yingzhu; Wu, Bingjie; Qin, Kun; Zhao, Jincun; Lou, Yongliang; Tan, Wenjie

2017-06-02

Although canine respiratory coronavirus (CRCoV) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of CRCoV (South Korea strain K37) has been obtained to date. Genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. Herein, we report a unique CRCoV strain, denoted strain BJ232, derived from a CRCoV-positive dog with a mild respiratory infection. Phylogenetic analysis based on complete genome of all available coronaviruses consistently show that CRCoV BJ232 is most closely related to human coronavirus OC43 (HCoV-OC43) and BCoV, forming a separate clade that split off early from other Betacoronavirus 1. Based on the phylogenetic and SimPlot analysis we propose that CRCoV-K37 was derived from genetic recombination between CRCoV-BJ232 and BCoV. In detail, spike (S) gene of CRCoV-K37 clustered with CRCoV-BJ232. However orf1ab, membrane (M) and nucleocapsid (N) genes were more related to Bovine coronavirus (BCoV) than CRCoV-B232. Molecular epidemic analysis confirmed the prevalence of CRCoV-BJ232 lineage around the world for a long time. Recombinant events among Betacoronavirus 1 may have implications for CRCoV transmissibility. All these findings provide further information regarding the origin of CRCoV. Copyright © 2017. Published by Elsevier B.V.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

OpenAIRE

Amer, Alazawy; Siti Suri, Arshad; Abdul Rahman, Omar; Mohd, Hair Bejo; Faruku, Bande; Saeed, Sharif; Tengku Azmi, Tengku Ibrahim

2012-01-01

Abstract Background Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first...

Cadherin complexes recruit mRNAs and RISC to regulate epithelial cell signaling.

Science.gov (United States)

Kourtidis, Antonis; Necela, Brian; Lin, Wan-Hsin; Lu, Ruifeng; Feathers, Ryan W; Asmann, Yan W; Thompson, E Aubrey; Anastasiadis, Panos Z

2017-10-02

Cumulative evidence demonstrates that most RNAs exhibit specific subcellular distribution. However, the mechanisms regulating this phenomenon and its functional consequences are still under investigation. Here, we reveal that cadherin complexes at the apical zonula adherens (ZA) of epithelial adherens junctions recruit the core components of the RNA-induced silencing complex (RISC) Ago2, GW182, and PABPC1, as well as a set of 522 messenger RNAs (mRNAs) and 28 mature microRNAs (miRNAs or miRs), via PLEKHA7. Top canonical pathways represented by these mRNAs include Wnt/β-catenin, TGF-β, and stem cell signaling. We specifically demonstrate the presence and silencing of MYC, JUN, and SOX2 mRNAs by miR-24 and miR-200c at the ZA. PLEKHA7 knockdown dissociates RISC from the ZA, decreases loading of the ZA-associated mRNAs and miRNAs to Ago2, and results in a corresponding increase of MYC, JUN, and SOX2 protein expression. The present work reveals a mechanism that directly links junction integrity to the silencing of a set of mRNAs that critically affect epithelial homeostasis. © 2017 Kourtidis et al.

Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency

OpenAIRE

Szczawinska‐Poplonyk, Aleksandra; Jonczyk‐Potoczna, Katarzyna; Breborowicz, Anna; Bartkowska‐Sniatkowska, Alicja; Figlerowicz, Magdalena

2012-01-01

Please cite this paper as: Szczawinska‐Poplonyk et al. (2012) Fatal respiratory distress syndrome due to coronavirus infection in a child with severe combined immunodeficiency. Influenza and Other Respiratory Viruses DOI: 10.1111/irv.12059. Coronaviruses have been demonstrated to contribute substantially to respiratory tract infections among the child population. Though infected children commonly present mild upper airway symptoms, in high‐risk patients with underlying conditions, particularl...

Cross host transmission in the emergence of MERS coronavirus

NARCIS (Netherlands)

C.B.E.M. Reusken (Chantal); V.S. Raj (Stalin); M.P.G. Koopmans D.V.M. (Marion); B.L. Haagmans (Bart)

2016-01-01

textabstractCoronaviruses (CoVs) able to infect humans emerge through cross-host transmission from animals. There is substantial evidence that the recent Middle East respiratory syndrome (MERS)-CoV outbreak is fueled by zoonotic transmission from dromedary camels. This is largely based on the fact

Conserved antigenic sites between MERS-CoV and Bat-coronavirus are revealed through sequence analysis.

Science.gov (United States)

Sharmin, Refat; Islam, Abul B M M K

2016-01-01

MERS-CoV is a newly emerged human coronavirus reported closely related with HKU4 and HKU5 Bat coronaviruses. Bat and MERS corona-viruses are structurally related. Therefore, it is of interest to estimate the degree of conserved antigenic sites among them. It is of importance to elucidate the shared antigenic-sites and extent of conservation between them to understand the evolutionary dynamics of MERS-CoV. Multiple sequence alignment of the spike (S), membrane (M), enveloped (E) and nucleocapsid (N) proteins was employed to identify the sequence conservation among MERS and Bat (HKU4, HKU5) coronaviruses. We used various in silico tools to predict the conserved antigenic sites. We found that MERS-CoV shared 30 % of its S protein antigenic sites with HKU4 and 70 % with HKU5 bat-CoV. Whereas 100 % of its E, M and N protein's antigenic sites are found to be conserved with those in HKU4 and HKU5. This sharing suggests that in case of pathogenicity MERS-CoV is more closely related to HKU5 bat-CoV than HKU4 bat-CoV. The conserved epitopes indicates their evolutionary relationship and ancestry of pathogenicity.

Detection by radioimmunoassay and enzyme-linked immunosorbent assay of coronavirus antibodies in bovine serum and lacteal secretions.

Science.gov (United States)

Rodak, L; Babiuk, L A; Acres, S D

1982-07-01

The sensitivity of a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA), and a serum neutralization assay (SN) for detecting antibodies to bovine coronavirus in serum and colostrum were compared. Although there proved to be a good correlation among all three assays (r = 0.915 and 0.964 for RIA with SN and ELISA, respectively), RIA and ELISA proved to be at least 10 times more sensitive than neutralization tests. By using these techniques, it was possible to detect a time-dependent decrease in antibody levels in bovine colostrum after parturition. Using ELISA, we demonstrated that 12 of 12 herds in Saskatchewan, and 109 of 110 animals tested, and antibody to bovine coronavirus. There was no elevated antibody response in serum or lacteal secretions of cows vaccinated once or twice with a commercially available modified live rota-coronavirus vaccine. In addition to being more sensitive than SN, ELISA and RIA proved to have other advantages for measuring antibody levels to bovine coronavirus and therefore warrant wider use as tools in diagnostic virology.

Crystallization and diffraction analysis of the SARS coronavirus nsp10–nsp16 complex

International Nuclear Information System (INIS)

Debarnot, Claire; Imbert, Isabelle; Ferron, François; Gluais, Laure; Varlet, Isabelle; Papageorgiou, Nicolas; Bouvet, Mickaël; Lescar, Julien; Decroly, Etienne; Canard, Bruno

2011-01-01

The expression, purification and crystallization of the SARS coronavirus nsp16 RNA-cap AdoMet-dependent (nucleoside-2′O)-methyltransferase in complex with its activating factor nsp10 are reported. To date, the SARS coronavirus is the only known highly pathogenic human coronavirus. In 2003, it was responsible for a large outbreak associated with a 10% fatality rate. This positive RNA virus encodes a large replicase polyprotein made up of 16 gene products (nsp1–16), amongst which two methyltransferases, nsp14 and nsp16, are involved in viral mRNA cap formation. The crystal structure of nsp16 is unknown. Nsp16 is an RNA-cap AdoMet-dependent (nucleoside-2′-O-)-methyltransferase that is only active in the presence of nsp10. In this paper, the expression, purification and crystallization of nsp10 in complex with nsp16 are reported. The crystals diffracted to a resolution of 1.9 Å resolution and crystal structure determination is in progress

Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition

International Nuclear Information System (INIS)

Versteeg, Gijs A.; Bredenbeek, Peter J.; Worm, Sjoerd H.E. van den; Spaan, Willy J.M.

2007-01-01

Many viruses encode antagonists to prevent interferon (IFN) induction. Infection of fibroblasts with the murine hepatitis coronavirus (MHV) and SARS-coronavirus (SARS-CoV) did not result in nuclear translocation of interferon-regulatory factor 3 (IRF3), a key transcription factor involved in IFN induction, and induction of IFN mRNA transcription. Furthermore, MHV and SARS-CoV infection could not prevent IFN induction by poly (I:C) or Sendai virus, suggesting that these CoVs do not inactivate IRF3-mediated transcription regulation, but apparently prevent detection of replicative RNA by cellular sensory molecules. Our data indicate that shielding of viral RNA to host cell sensors might be the main general mechanism for coronaviruses to prevent IFN induction

Coronavirus nucleocapsid proteins assemble constitutively in high molecular oligomers

NARCIS (Netherlands)

Cong, Yingying; Kriegenburg, Franziska; de Haan, Cornelis A. M.; Reggiori, Fulvio

2017-01-01

Coronaviruses (CoV) are enveloped viruses and rely on their nucleocapsid N protein to incorporate the positive-stranded genomic RNA into the virions. CoV N proteins form oligomers but the mechanism and relevance underlying their multimerization remain to be fully understood. Using in vitro pull-down

Crystal structure of Middle East respiratory syndrome coronavirus helicase.

Directory of Open Access Journals (Sweden)

Wei Hao

2017-06-01

Full Text Available Middle East respiratory syndrome coronavirus (MERS-CoV remains a threat to public health worldwide; however, effective vaccine or drug against CoVs remains unavailable. CoV helicase is one of the three evolutionary most conserved proteins in nidoviruses, thus making it an important target for drug development. We report here the first structure of full-length coronavirus helicase, MERS-CoV nsp13. MERS-CoV helicase has multiple domains, including an N-terminal Cys/His rich domain (CH with three zinc atoms, a beta-barrel domain and a C-terminal SF1 helicase core with two RecA-like subdomains. Our structural analyses show that while the domain organization of nsp13 is conserved throughout nidoviruses, the individual domains of nsp13 are closely related to the equivalent eukaryotic domains of Upf1 helicases. The most distinctive feature differentiating CoV helicases from eukaryotic Upf1 helicases is the interaction between CH domain and helicase core.

Human Coronaviruses: Insights into Environmental Resistance and Its Influence on the Development of New Antiseptic Strategies

Science.gov (United States)

Geller, Chloé; Varbanov, Mihayl; Duval, Raphaël E.

2012-01-01

The Coronaviridae family, an enveloped RNA virus family, and, more particularly, human coronaviruses (HCoV), were historically known to be responsible for a large portion of common colds and other upper respiratory tract infections. HCoV are now known to be involved in more serious respiratory diseases, i.e. bronchitis, bronchiolitis or pneumonia, especially in young children and neonates, elderly people and immunosuppressed patients. They have also been involved in nosocomial viral infections. In 2002–2003, the outbreak of severe acute respiratory syndrome (SARS), due to a newly discovered coronavirus, the SARS-associated coronavirus (SARS-CoV); led to a new awareness of the medical importance of the Coronaviridae family. This pathogen, responsible for an emerging disease in humans, with high risk of fatal outcome; underline the pressing need for new approaches to the management of the infection, and primarily to its prevention. Another interesting feature of coronaviruses is their potential environmental resistance, despite the accepted fragility of enveloped viruses. Indeed, several studies have described the ability of HCoVs (i.e. HCoV 229E, HCoV OC43 (also known as betacoronavirus 1), NL63, HKU1 or SARS-CoV) to survive in different environmental conditions (e.g. temperature and humidity), on different supports found in hospital settings such as aluminum, sterile sponges or latex surgical gloves or in biological fluids. Finally, taking into account the persisting lack of specific antiviral treatments (there is, in fact, no specific treatment available to fight coronaviruses infections), the Coronaviridae specificities (i.e. pathogenicity, potential environmental resistance) make them a challenging model for the development of efficient means of prevention, as an adapted antisepsis-disinfection, to prevent the environmental spread of such infective agents. This review will summarize current knowledge on the capacity of human coronaviruses to survive in the

Discovery of novel bat coronaviruses in south China that use the same receptor as MERS coronavirus.

Science.gov (United States)

Luo, Chu-Ming; Wang, Ning; Yang, Xing-Lou; Liu, Hai-Zhou; Zhang, Wei; Li, Bei; Hu, Ben; Peng, Cheng; Geng, Qi-Bin; Zhu, Guang-Jian; Li, Fang; Shi, Zheng-Li

2018-04-18

Middle East respiratory syndrome coronavirus (MERS-CoV) has represented a human health threat since 2012. Although several MERS-related CoVs, which belong to the same species as MERS-CoV, have been identified from bats, they do not use the MERS-CoV receptor, dipeptidyl peptidase 4 (DPP4). Here, we screened 1059 bat samples from at least 30 bat species collected in different regions in south China and identified 89 strains of lineage C betacoronaviruses, including Tylonycteris pachypus HKU4 , Pipistrellus pipistrellus HKU5, and MERS-related CoVs. We sequenced the full-length genomes of two positive samples collected from the great evening bat, Ia io , from Guangdong Province. The two genomes were highly similar and exhibited genomic structures identical to those of other lineage C betacoronaviruses. While they exhibited genome-wide nucleotide identities of only 75.3 to 81.2% with other MERS-related CoVs, their gene-coding regions were highly similar to their counterparts, except in the case of the spike proteins. Further protein--protein interaction assays demonstrated that the spike proteins of these MERS-related CoVs bind to the receptor DPP4. Recombination analysis suggested that the newly discovered MERS-related CoVs might have acquired their spike genes from a DPP4-recognizing bat HKU4. Our study provides further evidence that bats represent the evolutionary origins of MERS-CoV. IMPORTANCE Previous studies suggested that the Middle East respiratory syndrome coronavirus (MERS-CoV) may have originated in bats. However, its evolutionary path from bats to humans remains unclear. In this study, we discovered 89 novel lineage C betacoronaviruses (BetaCoVs) in eight bat species. We provide the evidence of a MERS-related CoV derived from the great evening bat that uses the same host receptor as human MERS-CoV. This virus also provides evidence for a natural recombination event between the bat MERS-related CoV and another bat coronavirus HKU4. Our study expands the host

Nuclear-Encoded Mitochondrial mRNAs: A Powerful Force in Axonal Growth and Development.

Science.gov (United States)

Gale, Jenna R; Aschrafi, Armaz; Gioio, Anthony E; Kaplan, Barry B

2018-04-01

Axons, their growth cones, and synaptic nerve terminals are neuronal subcompartments that have high energetic needs. As such, they are enriched in mitochondria, which supply the ATP necessary to meet these demands. To date, a heterogeneous population of nuclear-encoded mitochondrial mRNAs has been identified in distal axons and growth cones. Accumulating evidence suggests that the local translation of these mRNAs is required for mitochondrial maintenance and axonal viability. Here, we review evidence that suggests a critical role for axonal translation of nuclear-encoded mitochondrial mRNAs in axonal growth and development. Additionally, we explore the role that site-specific translation at the mitochondria itself may play in this process. Finally, we briefly review the clinical implications of dysregulation of local translation of mitochondrial-related mRNAs in neurodevelopmental disorders.

Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

Directory of Open Access Journals (Sweden)

Anna Lundin

2014-05-01

Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

W. Widagdo; L. Begeman (Lineke); D. Schipper (Debby); P.R.W.A. van Run (Peter); Cunningham, A.A. (Andrew A); Kley, N. (Nils); C.B.E.M. Reusken (Chantal); B.L. Haagmans (Bart); J.M.A. van den Brand (Judith)

2017-01-01

textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (DPP4) as its receptor.The distribution of DPP4 in the respiratory tract tissues of humans and camels reflects MERS-CoV tropism.Apart from

Tissue Distribution of the MERS-Coronavirus Receptor in Bats

NARCIS (Netherlands)

Widagdo, W; Begeman, Lineke; Schipper, Debby; van Run, Peter R; Cunningham, Andrew A; Kley, Nils; Reusken, Chantal B E M; Haagmans, Bart L; van den Brand, Judith M A

2017-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has been shown to infect both humans and dromedary camels using dipeptidyl peptidase-4 (DPP4) as its receptor. The distribution of DPP4 in the respiratory tract tissues of humans and camels reflects MERS-CoV tropism. Apart from dromedary

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

Science.gov (United States)

2012-01-01

Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

Identification and analysis of pig chimeric mRNAs using RNA sequencing data

Directory of Open Access Journals (Sweden)

Ma Lei

2012-08-01

Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

Diagnostic Methods for Feline Coronavirus: A Review

Directory of Open Access Journals (Sweden)

Saeed Sharif

2010-01-01

Full Text Available Feline coronaviruses (FCoVs are found throughout the world. Infection with FCoV can result in a diverse range of signs from clinically inapparent infections to a highly fatal disease called feline infectious peritonitis (FIP. FIP is one of the most serious viral diseases of cats. While there is neither an effective vaccine, nor a curative treatment for FIP, a diagnostic protocol for FCoV would greatly assist in the management and control of the virus. Clinical findings in FIP are non-specific and not helpful in making a differential diagnosis. Haematological and biochemical abnormalities in FIP cases are also non-specific. The currently available serological tests have low specificity and sensitivity for detection of active infection and cross-react with FCoV strains of low pathogenicity, the feline enteric coronaviruses (FECV. Reverse transcriptase polymerase chain reaction (RT-PCR has been used to detect FCoV and is rapid and sensitive, but results must be interpreted in the context of clinical findings. At present, a definitive diagnosis of FIP can be established only by histopathological examination of biopsies. This paper describes and compares diagnostic methods for FCoVs and includes a brief account of the virus biology, epidemiology, and pathogenesis.

Livestock Susceptibility to Infection with Middle East Respiratory Syndrome Coronavirus

NARCIS (Netherlands)

Vergara-Alert, Júlia; van den Brand, Judith M A; Widagdo, W; Muñoz, Marta; Raj, V Stalin; Schipper, Debby; Solanes, David; Cordón, Ivan; Bensaid, Albert; Haagmans, Bart L; Segalés, Joaquim

Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs.

Feline coronavirus: Insights into viral pathogenesis based on the spike protein structure and function.

Science.gov (United States)

Jaimes, Javier A; Whittaker, Gary R

2018-04-01

Feline coronavirus (FCoV) is an etiological agent that causes a benign enteric illness and the fatal systemic disease feline infectious peritonitis (FIP). The FCoV spike (S) protein is considered the viral regulator for binding and entry to the cell. This protein is also involved in FCoV tropism and virulence, as well as in the switch from enteric disease to FIP. This regulation is carried out by spike's major functions: receptor binding and virus-cell membrane fusion. In this review, we address important aspects in FCoV genetics, replication and pathogenesis, focusing on the role of S. To better understand this, FCoV S protein models were constructed, based on the human coronavirus NL63 (HCoV-NL63) S structure. We describe the specific structural characteristics of the FCoV S, in comparison with other coronavirus spikes. We also revise the biochemical events needed for FCoV S activation and its relation to the structural features of the protein. Copyright © 2018 Elsevier Inc. All rights reserved.

Characterization of mTOR-Responsive Truncated mRNAs in Cell Proliferation

Science.gov (United States)

2017-07-01

These findings identify a previously uncharacterized role for mTOR in modulating 3’- UTR length of mRNAs by alternative polyadenylation ( APA ). Another...outcome of APA in the mTOR-activated transcriptome is an early termination of mRNA transcription to produce truncated mRNAs with polyadenylation in...for exhaustive analysis of Alternative cleavage and polyadenylation ( APA ) events (Figure 1). In IntMAP, first the position of multiple

The race to decipher the top secrets of TOP mRNAs.

Science.gov (United States)

Meyuhas, Oded; Kahan, Tamar

2015-07-01

Cells encountering hostile growth conditions, like those residing in the middle of a newly developing solid tumor, conserve resources and energy by downregulating protein synthesis. One mechanism in this response is the translational repression of multiple mRNAs that encode components of the translational apparatus. This coordinated translational control is carried through a common cis-regulatory element, the 5' Terminal OligoPyrimidine motif (5'TOP), after which these mRNAs are referred to as TOP mRNAs. Subsequent to the initial structural and functional characterization of members of this family, the research of TOP mRNAs has progressed in three major directions: a) delineating the landscape of the family; b) establishing the pathways that transduce stress cues into selective translational repression; and c) attempting to decipher the most proximal trans-acting factor(s) and defining its mode of action--a repressor or activator. The present chapter critically reviews the development in these three avenues of research with a special emphasis on the two "top secrets" of the TOP mRNA family: the scope of its members and the identity of the proximal cellular regulator(s). This article is part of a Special Issue entitled: Translation and Cancer. Copyright © 2014 Elsevier B.V. All rights reserved.

Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences

Czech Academy of Sciences Publication Activity Database

Drexler, J. F.; Gloza-Rausch, F.; Glende, J.; Corman, V. M.; Muth, D.; Goettsche, M.; Seebens, A.; Niedrig, M.; Pfefferle, S.; Yordanov, S.; Zhelyazkov, L.; Hermanns, U.; Vallo, Peter; Lukashev, A.; Müller, M. A.; Deng, H.; Herrler, G.; Drosten, C.

2010-01-01

Roč. 84, č. 21 (2010), s. 11336-11349 ISSN 0022-538X Institutional research plan: CEZ:AV0Z60930519 Keywords : cross-species transmission * SARS-like coronavirus es * reservoir hosts * horseshoe bats Subject RIV: EE - Microbiology, Virology Impact factor: 5.189, year: 2010

MERS Coronavirus Neutralizing Antibodies in Camels, Eastern Africa, 1983-1997

NARCIS (Netherlands)

Müller, Marcel A; Corman, Victor Max; Jores, Joerg; Meyer, Benjamin; Younan, Mario; Liljander, Anne; Bosch, Berend-Jan; Lattwein, Erik; Hilali, Mosaad; Musa, Bakri E; Bornstein, Set; Drosten, Christian

2014-01-01

To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main

Vacuolating encephalitis in mice infected by human coronavirus OC43

International Nuclear Information System (INIS)

Jacomy, Helene; Talbot, Pierre J.

2003-01-01

Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals

First full length sequences of the S gene of European isolates reveal further diversity among turkey coronaviruses.

OpenAIRE

2011-01-01

Abstract An increasing incidence of enteric disorders clinically evocative of the poult enteritis complex has been observed in turkeys in France since 2003. Using a newly designed real-time RT-PCR assay specific for the nucleocapsid (N) gene of infectious bronchitis virus (IBV) and turkey coronaviruses (TCoV), coronaviruses were identified in 37 % of the intestinal samples collected from diseased turkey flocks. The full length Spike (S) gene of these viruses was amplified, cloned a...

Lack of association between infection with a novel human coronavirus (HCoV), HCoV-NH, and Kawasaki disease in Taiwan

NARCIS (Netherlands)

Chang, Luan-Yin; Chiang, Bor-Luen; Kao, Chuan-Liang; Wu, Mei-Hwan; Chen, Pei-Jer; Berkhout, Ben; Yang, Hui-Ching; Huang, Li-Min

2006-01-01

We investigated whether infection with a novel human coronavirus (HCoV), called "New Haven coronavirus" (HCoV-NH)--which is similar to and likely represents the same species as another novel HCoV, HCoV-NL63--is associated with Kawasaki disease (KD) in Taiwan. Fifty-three patients with KD were

Cleavage of group 1 coronavirus spike proteins: how furin cleavage is traded off against heparan sulfate binding upon cell culture adaptation

NARCIS (Netherlands)

Haan, de C.A.M.; Haijema, B.J.; Schellen, P.; Wichgers Schreur, P.J.; Lintelo, te E.; Vennema, H.; Rottier, P.J.M.

2008-01-01

A longstanding enigmatic feature of the group 1 coronaviruses is the uncleaved phenotype of their spike protein, an exceptional property among class I fusion proteins. Here, however, we show that some group 1 coronavirus spike proteins carry a furin enzyme recognition motif and can actually be

Structure and inhibition of the SARS coronavirus envelope protein ion channel.

Directory of Open Access Journals (Sweden)

Konstantin Pervushin

2009-07-01

Full Text Available The envelope (E protein from coronaviruses is a small polypeptide that contains at least one alpha-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA, but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV that the transmembrane domain of E protein (ETM forms pentameric alpha-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular alpha-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293 cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA, but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.

Stage-related expression of mRNAs during pollen development in lily and tobacco.

Science.gov (United States)

Schrauwen, J A; de Groot, P F; van Herpen, M M; van der Lee, T; Reynen, W H; Weterings, K A; Wullems, G J

1990-09-01

Homogeneous populations of developing microspores and pollen from anthers of lily (Lilium longiflorum Thumb.) and tobacco (Nicotiana tabacum L.) show a continuous production of biomass, reaching a maximum in young pollen. The rate of RNA synthesis was 460 fg · h(-1) in young binucleate cells, 138 fg · h(-1) in late binucleate cells and 56 fg · h(-1) in microspores. The mRNA population in developing pollen can be separated into three groups. In the first group, certain types of mRNAs are present at a constant level during all stages of development. A second group is characteristic of young pollen and increases quantitatively until anthesis. A third group is seen transiently; to this belong mRNAs present only before mitosis or at a distinct cell stage after mitosis. Some of the translation products of this latter group of mRNAs showed similarities between lily and tobacco on two-dimensional gels in respect of molecular weight and isolectric point, indicating that those mRNAs and proteins play a role in the regulation of pollen development.

Screening of an FDA-approved compound library identifies four small-molecule inhibitors of Middle East respiratory syndrome coronavirus replication in cell culture

NARCIS (Netherlands)

A.H. de Wilde (Adriaan); D. Jochmans (Dirk); C.C. Posthuma (Clara); J.C. Zevenhoven-Dobbe (Jessika); S. van Nieuwkoop (Stefan); T.M. Bestebroer (Theo); B.G. van den Hoogen (Bernadette); J. Neyts; E.J. Snijder (Eric)

2014-01-01

textabstractCoronaviruses can cause respiratory and enteric disease in a wide variety of human and animal hosts. The 2003 outbreak of severe acute respiratory syndrome (SARS) first demonstrated the potentially lethal consequences of zoonotic coronavirus infections in humans. In 2012, a similar

Selective translational repression of truncated proteins from frameshift mutation-derived mRNAs in tumors.

Directory of Open Access Journals (Sweden)

Kwon Tae You

2007-05-01

Full Text Available Frameshift and nonsense mutations are common in tumors with microsatellite instability, and mRNAs from these mutated genes have premature termination codons (PTCs. Abnormal mRNAs containing PTCs are normally degraded by the nonsense-mediated mRNA decay (NMD system. However, PTCs located within 50-55 nucleotides of the last exon-exon junction are not recognized by NMD (NMD-irrelevant, and some PTC-containing mRNAs can escape from the NMD system (NMD-escape. We investigated protein expression from NMD-irrelevant and NMD-escape PTC-containing mRNAs by Western blotting and transfection assays. We demonstrated that transfection of NMD-irrelevant PTC-containing genomic DNA of MARCKS generates truncated protein. In contrast, NMD-escape PTC-containing versions of hMSH3 and TGFBR2 generate normal levels of mRNA, but do not generate detectable levels of protein. Transfection of NMD-escape mutant TGFBR2 genomic DNA failed to generate expression of truncated proteins, whereas transfection of wild-type TGFBR2 genomic DNA or mutant PTC-containing TGFBR2 cDNA generated expression of wild-type protein and truncated protein, respectively. Our findings suggest a novel mechanism of gene expression regulation for PTC-containing mRNAs in which the deleterious transcripts are regulated either by NMD or translational repression.

Clinico-epidemiological characteristics of acute respiratory infections caused by coronavirus OC43, NL63 and 229E.

Science.gov (United States)

Reina, J; López-Causapé, C; Rojo-Molinero, E; Rubio, R

2014-12-01

Acute respiratory infection is a very common condition in the general population. The majority of these infections are due to viruses. This study attempted to determine the clinical and epidemiological characteristics of adult patients with respiratory infection by the coronavirus OC43, NL63 and 229E. Between January 2013 and February 2014, we prospectively studied all patients with suspected clinical respiratory infection by taking throat swabs and performing a reverse transcription polymerase chain reaction in search of coronavirus. In 48 cases (7.0% of the 686 enrolled patients; 12.6% of the 381 in whom a virus was detected) the presence of a coronavirus demonstrated. In 24 cases, the virus was OC43 (50%); in 14 cases, the virus was NL63 (29%); and in 10 cases, the virus was 229E (21%). The mean age was 54.5 years, with a slight predominance of men. The most common clinical presentations were nonspecific influenza symptoms (43.7%), pneumonia (29.2%) and chronic obstructive pulmonary disease exacerbation (8.3%). Fifty-two percent of the patients required hospitalization, and 2 patients required intensive care. There were no deaths. Acute respiratory infections caused by coronavirus mainly affect middle-aged male smokers, who are often affected by previous diseases. The most common clinical picture has been nonspecific influenza symptoms. Copyright © 2014 Elsevier España, S.L.U. All rights reserved.

Sequestration of highly expressed mRNAs in cytoplasmic granules, P-bodies, and stress granules enhances cell viability.

Directory of Open Access Journals (Sweden)

Anna Lavut

Full Text Available Transcriptome analyses indicate that a core 10%-15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical

Translational coregulation of 5′TOP mRNAs by TIA-1 and TIAR

Science.gov (United States)

Damgaard, Christian Kroun; Lykke-Andersen, Jens

2011-01-01

The response of cells to changes in their environment often requires coregulation of gene networks, but little is known about how this can occur at the post-transcriptional level. An important example of post-transcriptional coregulation is the selective translational regulation in response to growth conditions of mammalian mRNAs that encode protein biosynthesis factors and contain hallmark 5′-terminal oligopyrimidine tracts (5′TOP). However, the responsible trans-factors and the mechanism by which they coregulate 5′TOP mRNAs have remained elusive. Here we identify stress granule-associated TIA-1 and TIAR proteins as key factors in human 5′TOP mRNA regulation, which upon amino acid starvation assemble onto the 5′ end of 5′TOP mRNAs and arrest translation at the initiation step, as evidenced by TIA-1/TIAR-dependent 5′TOP mRNA translation repression, polysome release, and accumulation in stress granules. This requires starvation-mediated activation of the GCN2 (general control nonderepressible 2) kinase and inactivation of the mTOR (mammalian target of rapamycin) signaling pathway. Our findings provide a mechanistic explanation to the long-standing question of how the network of 5′TOP mRNAs are coregulated according to amino acid availability, thereby allowing redirection of limited resources to mount a nutrient deprivation response. This presents a fundamental example of how a group of mRNAs can be translationally coregulated in response to changes in the cellular environment. PMID:21979918

Middle East respiratory syndrome coronavirus in dromedary camels: An outbreak investigation

NARCIS (Netherlands)

B.L. Haagmans (Bart); S.H.S. Al Dhahiry (Said); C.B.E.M. Reusken (Chantal); V.S. Raj (Stalin); M. Galiano (Monica); R.H. Myers (Richard); G-J. Godeke (Gert-Jan); M. Jonges (Marcel); E. Farag (Elmoubasher); A. Diab (Ayman); H. Ghobashy (Hazem); F. Alhajri (Farhoud); M. Al-Thani (Mohamed); S.A. Al-Marri (Salih); H.E. Al Romaihi (Hamad); A. Al Khal (Abdullatif); A. Bermingham (Alison); A.D.M.E. Osterhaus (Albert); M.M. AlHajri (Mohd); M.P.G. Koopmans D.V.M. (Marion)

2014-01-01

textabstractBackground: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe lower respiratory tract infection in people. Previous studies suggested dromedary camels were a reservoir for this virus. We tested for the presence of MERS-CoV in dromedary camels from a farm in Qatar

Coronavirus in Pigs: Significance and Presentation of Swine Epidemic Diarrhea Virus (PEDV in Colombia

Directory of Open Access Journals (Sweden)

Ricardo Piñeros

2015-05-01

Full Text Available The article seeks to study general aspects of the main coronaviruses affecting pigs, their presentation in Colombia, and particular aspects of porcine epidemic diarrhea virus (PEDV, emerging in different countries and generating a great impact on the health and economy of the swine industry. The main coronaviruses affecting swine are porcine transmissible gastroenteritis virus (TGEV, porcine respiratory coronavirus (PRCV, porcine hemagglutinating encephalomyelitis virus (PHEV, PEDV, and porcine deltacoronavirus (PDCoV. Long ago in Colombia there had been reports of TGEV and PRCV associated with the importation of animals from the United States, which was controlled in the infected farms and in quarantine units. PEDV was first detected in Colombia in mid-March 2014; the Colombian Agricultural Institute issued a health alert in Neiva (Huila, Fusagasugá and Silvania (Cundinamarca, and Puerto López (Meta due to the unusual presentation of epidemic vomiting and diarrhea in young and adult animals, abortion in pregnant sows, with high mortality rates (up to 100% in animals during the first week of age. At present the disease has been reported in other municipalities of the country as well as in different countries with similar clinical conditions and mortality rates in pigs with high economic losses for the swine sector.

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

NARCIS (Netherlands)

P.A. Rota (Paul); M.S. Oberste (Steven); S.S. Monroe (Stephan); W.A. Nix (Allan); R. Campagnoli (Ray); J.P. Icenogle (Joseph); S. Penaranda; B. Bankamp (Bettina); K. Maher (Kaija); M.H. Chen (Min-hsin); S. Tong (Suxiong); A. Tamin (Azaibi); L. Lowe (Luis); M. Frace (Michael); J.L. DeRisi (Joseph); Q. Chen (Qi); D. Wang (David); D.D. Erdman (Dean); T.C. Peret (Teresa); C. Burns (Cara); T.G. Ksiazek (Thomas); P.E. Rollin (Pierre); A. Sanchez (Berenguer); S. Liffick (Stephanie); B. Holloway (Brian); J. Limor (Josef); K. McCaustland (Karen); M. Olsen-Rasmussen (Mellissa); S. Gunther; A.D.M.E. Osterhaus (Albert); C. Drosten (Christian); M.A. Pallansch (Mark); L.J. Anderson (Larry); W.J. Belline; R.A.M. Fouchier (Ron)

2003-01-01

textabstractIn March 2003, a novel coronavirus (SARS-CoV) was discovered in association with cases of severe acute respiratory syndrome (SARS). The sequence of the complete genome of SARS-CoV was determined, and the initial characterization of the viral genome is presented in this report. The

A rare cause of acute flaccid paralysis: Human coronaviruses

OpenAIRE

Turgay, Cokyaman; Emine, Tekin; Ozlem, Koken; Muhammet, S. Paksu; Haydar, A. Tasdemir

2015-01-01

Acute flaccid paralysis (AFP) is a life-threatening clinical entity characterized by weakness in the whole body muscles often accompanied by respiratory and bulbar paralysis. The most common cause is Gullian-Barre syndrome, but infections, spinal cord diseases, neuromuscular diseases such as myasthenia gravis, drugs and toxins, periodic hypokalemic paralysis, electrolyte disturbances, and botulism should be considered as in the differential diagnosis. Human coronaviruses (HCoVs) cause common ...

Severe acute respiratory syndrome--a new coronavirus from the Chinese dragon's lair

DEFF Research Database (Denmark)

Knudsen, T B; Kledal, T N; Andersen, O

2003-01-01

current worldwide distribution. The concerted efforts of a globally united scientific community have led to the independent isolation and identification of a novel coronavirus from SARS patients by several groups. The extraordinarily rapid isolation of a causative agent of this newly emerged infectious...

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

Directory of Open Access Journals (Sweden)

Markus Hoffmann

Full Text Available 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.

Middle East Respiratory Syndrome (MERS) coronavirus seroprevalence in domestic livestock in Saudi Arabia, 2010 to 2013.

Science.gov (United States)

Hemida, M G; Perera, R A; Wang, P; Alhammadi, M A; Siu, L Y; Li, M; Poon, L L; Saif, L; Alnaeem, A; Peiris, M

2013-12-12

In Saudi Arabia, including regions of Riyadh and Al Ahsa, pseudoparticle neutralisation (ppNT) and microneutralisation (MNT) tests detected no antibodies to Middle East Respiratory Syndrome coronavirus (MERS-CoV) in sheep (n= 100), goats (n= 45), cattle (n= 50) and chickens (n= 240). Dromedary camels however, had a high prevalence of MERS-CoV antibodies. Bovine coronavirus (BCoV) infected sera from cattle had no cross-reactivity in MERS-CoV ppNT or MNT, while many dromedary camels’ sera reacted to both BCoV and MERS-CoV. Some nevertheless displayed specific serologic reaction profiles to MERS-CoV.

Accumulation of long-lived mRNAs associated with germination in embryos during seed development of rice

Science.gov (United States)

Sano, Naoto; Ono, Hanako; Murata, Kazumasa; Yamada, Tetsuya; Hirasawa, Tadashi; Kanekatsu, Motoki

2015-01-01

Mature dry seeds contain translatable mRNAs called long-lived mRNAs. Early studies have shown that protein synthesis during the initial phase of seed germination occurs from long-lived mRNAs, without de novo transcription. However, the gene expression systems that generate long-lived mRNAs in seeds are not well understood. To examine the accumulation of long-lived mRNAs in developing rice embryos, germination tests using the transcriptional inhibitor actinomycin D (Act D) were performed with the Japonica rice cultivar Nipponbare. Although over 70% of embryos at 10 days after flowering (DAF) germinated in the absence of the inhibitor, germination was remarkably impaired in embryos treated with Act D. In contrast, more than 70% of embryos at 20, 25, 30 and 40 DAF germinated in the presence of Act D. The same results were obtained when another cultivar, Koshihikari, was used, indicating that the long-lived mRNAs required for germination predominantly accumulate in embryos between 10 and 20 DAF during seed development. RNA-Seq identified 529 long-lived mRNA candidates, encoding proteins such as ABA, calcium ion and phospholipid signalling-related proteins, and HSP DNA J, increased from 10 to 20 DAF and were highly abundant in 40 DAF embryos of Nipponbare and Koshihikari. We also revealed that these long-lived mRNA candidates are clearly up-regulated in 10 DAF germinating embryos after imbibition, suggesting that the accumulation of these mRNAs in embryos is indispensable for the induction of germination. The findings presented here may facilitate in overcoming irregular seed germination or producing more vigorous seedlings. PMID:25941326

Feline Coronaviruses: Pathogenesis of Feline Infectious Peritonitis.

Science.gov (United States)

Tekes, G; Thiel, H-J

2016-01-01

Feline infectious peritonitis (FIP) belongs to the few animal virus diseases in which, in the course of a generally harmless persistent infection, a virus acquires a small number of mutations that fundamentally change its pathogenicity, invariably resulting in a fatal outcome. The causative agent of this deadly disease, feline infectious peritonitis virus (FIPV), arises from feline enteric coronavirus (FECV). The review summarizes our current knowledge of the genome and proteome of feline coronaviruses (FCoVs), focusing on the viral surface (spike) protein S and the five accessory proteins. We also review the current classification of FCoVs into distinct serotypes and biotypes, cellular receptors of FCoVs and their presumed role in viral virulence, and discuss other aspects of FIPV-induced pathogenesis. Our current knowledge of genetic differences between FECVs and FIPVs has been mainly based on comparative sequence analyses that revealed "discriminatory" mutations that are present in FIPVs but not in FECVs. Most of these mutations result in amino acid substitutions in the S protein and these may have a critical role in the switch from FECV to FIPV. In most cases, the precise roles of these mutations in the molecular pathogenesis of FIP have not been tested experimentally in the natural host, mainly due to the lack of suitable experimental tools including genetically engineered virus mutants. We discuss the recent progress in the development of FCoV reverse genetics systems suitable to generate recombinant field viruses containing appropriate mutations for in vivo studies. © 2016 Elsevier Inc. All rights reserved.

Antibodies against MERS coronavirus in dromedaries, United Arab Emirates, 2003 and 2013

NARCIS (Netherlands)

Meyer, Benjamin; Müller, Marcel A.; Corman, Victor M.; Reusken, Chantal B E M; Ritz, Daniel; Godeke, Gert Jan; Lattwein, Erik; Kallies, Stephan; Siemens, Artem; van Beek, Janko; Drexler, Jan F.; Muth, Doreen; Bosch, Berend Jan; Wernery, Ulrich; Koopmans, Marion P G; Wernery, Renate; Drosten, Christian

2014-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary

Qualitative and quantitative ultrastructural analysis of the membrane rearrangements induced by coronavirus

NARCIS (Netherlands)

Ulasli, M.; Verheije, M.H.; de Haan, C.A.M.; Reggiori, F.M.

2011-01-01

Coronaviruses (CoV) are enveloped positive-strand RNA viruses that induce different membrane rearrangements in infected cells in order to efficiently replicate and assemble. The origin, the protein composition and the function of these structures are not well established. To shed further light on

A Structural analysis of M protein in coronavirus assembly and morphology

DEFF Research Database (Denmark)

W. Neuman, Benjamin; Kiss, Gabriella; H. Kunding, Andreas

2011-01-01

The M protein of coronavirus plays a central role in virus assembly, turning cellular membranes into workshops where virus and host factors come together to make new virus particles. We investigated how M structure and organization is related to virus shape and size using cryo-electron microscopy...... protein functions to promote virus assembly....

The Severe Acute Respiratory Syndrome (SARS-coronavirus 3a protein may function as a modulator of the trafficking properties of the spike protein

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Tan Yee-Joo

2005-02-01

Full Text Available Abstract Background A recent publication reported that a tyrosine-dependent sorting signal, present in cytoplasmic tail of the spike protein of most coronaviruses, mediates the intracellular retention of the spike protein. This motif is missing from the spike protein of the severe acute respiratory syndrome-coronavirus (SARS-CoV, resulting in high level of surface expression of the spike protein when it is expressed on its own in vitro. Presentation of the hypothesis It has been shown that the severe acute respiratory syndrome-coronavirus genome contains open reading frames that encode for proteins with no homologue in other coronaviruses. One of them is the 3a protein, which is expressed during infection in vitro and in vivo. The 3a protein, which contains a tyrosine-dependent sorting signal in its cytoplasmic domain, is expressed on the cell surface and can undergo internalization. In addition, 3a can bind to the spike protein and through this interaction, it may be able to cause the spike protein to become internalized, resulting in a decrease in its surface expression. Testing the hypothesis The effects of 3a on the internalization of cell surface spike protein can be examined biochemically and the significance of the interplay between these two viral proteins during viral infection can be studied using reverse genetics methodology. Implication of the hypothesis If this hypothesis is proven, it will indicate that the severe acute respiratory syndrome-coronavirus modulates the surface expression of the spike protein via a different mechanism from other coronaviruses. The interaction between 3a and S, which are expressed from separate subgenomic RNA, would be important for controlling the trafficking properties of S. The cell surface expression of S in infected cells significantly impacts viral assembly, viral spread and viral pathogenesis. Modulation by this unique pathway could confer certain advantages during the replication of the severe

The role of viral population diversity in adaptation of bovine coronavirus to new host environments.

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Monica K Borucki

Full Text Available The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing and 38,000×(Illumina. The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were "selected" from a pre-existing pool rather than through de novo mutation and subsequent population fixation.

Granules Harboring Translationally Active mRNAs Provide a Platform for P-Body Formation following Stress

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Jennifer Lui

2014-11-01

Full Text Available The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

Acute and chronic glucocorticoid treatments regulate astrocyte-enriched mRNAs in multiple brain regions in vivo

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Bradley S. Carter

2013-08-01

Full Text Available Previous studies have primarily interpreted gene expression regulation by glucocorticoids in the brain in terms of impact on neurons; however, less is known about the corresponding impact of glucocorticoids on glia and specifically astrocytes in vivo. Recent microarray experiments have identified glucocorticoid-sensitive mRNAs in primary astrocyte cell culture, including a number of mRNAs that have reported astrocyte-enriched expression patterns relative to other brain cell types. Here, we have tested whether elevations of glucocorticoids regulate a subset of these mRNAs in vivo following acute and chronic corticosterone exposure in adult mice. Acute corticosterone exposure was achieved by a single injection of 10 mg/kg corticosterone, and tissue samples were harvested two hours post-injection. Chronic corticosterone exposure was achieved by administering 10 mg/mL corticosterone via drinking water for two weeks. Gene expression was then assessed in two brain regions associated with glucocorticoid action (prefrontal cortex and hippocampus by qPCR and by in situ hybridization. The majority of measured mRNAs regulated by glucocorticoids in astrocytes in vitro were similarly regulated by acute and/or chronic glucocorticoid exposure in vivo. In addition, the expression levels for mRNAs regulated in at least one corticosterone exposure condition (acute/chronic demonstrated moderate positive correlation between the two conditions by brain region. In situ hybridization analyses suggest that select mRNAs are regulated by chronic corticosterone exposure specifically in astroctyes based on (1 similar general expression patterns between corticosterone-treated and vehicle-treated animals and (2 similar expression patterns to the pan-astrocyte marker Aldh1l1. Our findings demonstrate that glucocorticoids regulate astrocyte-enriched mRNAs in vivo and suggest that glucocorticoids regulate gene expression in the brain in a cell type-dependent fashion.

Middle East Respiratory Syndrome Coronavirus Antibodies in Dromedary Camels, Bangladesh, 2015

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Islam, Ariful; Rostal, Melinda K.; Islam, Shariful; Rahman, Mohammed Ziaur; Hossain, Mohammed Enayet; Uzzaman, Mohammed Salim; Munster, Vincent J.; Peiris, Malik; Flora, Meerjady Sabrina; Rahman, Mahmudur; Daszak, Peter

2018-01-01

Dromedary camels are bred domestically and imported into Bangladesh. In 2015, of 55 camels tested for Middle East respiratory syndrome coronavirus in Dhaka, 17 (31%) were seropositive, including 1 bred locally. None were PCR positive. The potential for infected camels in urban markets could have public health implications and warrants further investigation. PMID:29664373

SARS-Coronavirus ancestor's foot-prints in South-East Asian bat colonies and the refuge theory.

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Gouilh, Meriadeg Ar; Puechmaille, Sébastien J; Gonzalez, Jean-Paul; Teeling, Emma; Kittayapong, Pattamaporn; Manuguerra, Jean-Claude

2011-10-01

One of the great challenges in the ecology of infectious diseases is to understand what drives the emergence of new pathogens including the relationship between viruses and their hosts. In the case of the emergence of SevereAcute Respiratory Syndrome Coronavirus (SARS-CoV), several studies have shown coronavirus diversity in bats as well as the existence of SARS-CoV infection in apparently healthy bats, suggesting that bats may be a crucial host in the genesis of this disease. To elucidate the biogeographic origin of SARS-CoV and investigate the role that bats played in its emergence, we amplified coronavirus sequences from bat species captured throughout Thailand and assessed the phylogenetic relationships to each other and to other published coronavirus sequences. To this end, RdRp sequence of Coronavirinae was targeted by RT-PCR in non-invasive samples from bats collected in Thailand. Two new coronaviruses were detected in two bat species: one Betacoronavirus in Hipposideros larvatus and one Alphacoronavirus in Hipposiderosarmiger. Interestingly, these viruses from South-East Asia are related to those previously detected in Africa (Betacoronavirus-b) or in Europe (Alphacoronavirus & Betacoronavirus-b). These findings illuminate the origin and the evolutionary history of the SARS-CoV group found in bats by pushing forward the hypothesis of a Betacoronavirus spill-over from Hipposideridae to Rhinolophidae and then from Rhinolophidae to civets and Human. All reported Betacoronaviruses-b (SARS-CoV group) of Hipposideridae and Rhinolophidae respectively cluster in two groups despite their broad geographic distribution and the sympatry of their hosts, which is in favor of an ancient and genetically independent evolution of Betacoronavirus-b clusters in these families. Moreover, despite its probable pathogenicity, we found that a Betacoronavirus-b can persistently infect a medium-sized hipposiderid bat colony. These findings illustrate the importance of the host

Searching for animal models and potential target species for emerging pathogens: Experience gained from Middle East respiratory syndrome (MERS coronavirus

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Júlia Vergara-Alert

2017-06-01

Full Text Available Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013–2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in 2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV, which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV, associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species in the transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed.

Axonal and dendritic localization of mRNAs for glycogen-metabolizing enzymes in cultured rodent neurons.

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Pfeiffer-Guglielmi, Brigitte; Dombert, Benjamin; Jablonka, Sibylle; Hausherr, Vanessa; van Thriel, Christoph; Schöbel, Nicole; Jansen, Ralf-Peter

2014-06-04

Localization of mRNAs encoding cytoskeletal or signaling proteins to neuronal processes is known to contribute to axon growth, synaptic differentiation and plasticity. In addition, a still increasing spectrum of mRNAs has been demonstrated to be localized under different conditions and developing stages thus reflecting a highly regulated mechanism and a role of mRNA localization in a broad range of cellular processes. Applying fluorescence in-situ-hybridization with specific riboprobes on cultured neurons and nervous tissue sections, we investigated whether the mRNAs for two metabolic enzymes, namely glycogen synthase (GS) and glycogen phosphorylase (GP), the key enzymes of glycogen metabolism, may also be targeted to neuronal processes. If it were so, this might contribute to clarify the so far enigmatic role of neuronal glycogen. We found that the mRNAs for both enzymes are localized to axonal and dendritic processes in cultured lumbar spinal motoneurons, but not in cultured trigeminal neurons. In cultured cortical neurons which do not store glycogen but nevertheless express glycogen synthase, the GS mRNA is also subject to axonal and dendritic localization. In spinal motoneurons and trigeminal neurons in situ, however, the mRNAs could only be demonstrated in the neuronal somata but not in the nerves. We could demonstrate that the mRNAs for major enzymes of neural energy metabolism can be localized to neuronal processes. The heterogeneous pattern of mRNA localization in different culture types and developmental stages stresses that mRNA localization is a versatile mechanism for the fine-tuning of cellular events. Our findings suggest that mRNA localization for enzymes of glycogen metabolism could allow adaptation to spatial and temporal energy demands in neuronal events like growth, repair and synaptic transmission.

Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

International Nuclear Information System (INIS)

Shakin, S.H.; Liebhaber, S.A.

1987-01-01

The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

Single-molecule imaging of {beta}-actin mRNAs in the cytoplasm of a living cell

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Yamagishi, Mai; Ishihama, Yo [Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shirasaki, Yoshitaka [Laboratory of Genome Technology, Department of Human Genome Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818 (Japan); Kurama, Hideki [Major in Integrative Bioscience and Biomedical Engineering, Graduate School of Science and Engineering, Waseda University, Shinjuku-ku, Tokyo 169-8555 (Japan); Funatsu, Takashi, E-mail: funatsu@mail.ecc.u-tokyo.ac.jp [Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033 (Japan); Center for NanoBio Integration, The University of Tokyo, Bunkyo-ku, Tokyo 113-8656 (Japan)

2009-04-15

{beta}-Actin mRNA labeled with an MS2-EGFP fusion protein was expressed in chicken embryo fibroblasts and its localization and movement were analyzed by single-molecule imaging. Most {beta}-Actin mRNAs localized to the leading edge, while some others were observed in the perinuclear region. Singe-molecule tracking of individual mRNAs revealed that the majority of mRNAs were in unrestricted Brownian motion at the leading edge and in restricted Brownian motion in the perinuclear region. The macroscopic diffusion coefficient of mRNA (D{sub MACRO}) at the leading edge was 0.3 {mu}m{sup 2}/s. On the other hand, D{sub MACRO} in the perinuclear region was 0.02 {mu}m{sup 2}/s. The destruction of microfilaments with cytochalasin D, which is known to delocalize {beta}-actin mRNAs, led to an increase in D{sub MACRO} to 0.2 {mu}m{sup 2}/s in the perinuclear region. These results suggest that the microstructure, composed of microfilaments, serves as a barrier for the movement of {beta}-actin mRNA.

A novel pancoronavirus RT-PCR assay: frequent detection of human coronavirus NL63 in children hospitalized with respiratory tract infections in Belgium

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Berkhout Ben

2005-02-01

Full Text Available Abstract Background Four human coronaviruses are currently known to infect the respiratory tract: human coronaviruses OC43 (HCoV-OC43 and 229E (HCoV-229E, SARS associated coronavirus (SARS-CoV and the recently identified human coronavirus NL63 (HCoV-NL63. In this study we explored the incidence of HCoV-NL63 infection in children diagnosed with respiratory tract infections in Belgium. Methods Samples from children hospitalized with respiratory diseases during the winter seasons of 2003 and 2004 were evaluated for the presence of HCoV-NL63 using a optimized pancoronavirus RT-PCR assay. Results Seven HCoV-NL63 positive samples were identified, six were collected during January/February 2003 and one at the end of February 2004. Conclusions Our results support the notation that HCoV-NL63 can cause serious respiratory symptoms in children. Sequence analysis of the S gene showed that our isolates could be classified into two subtypes corresponding to the two prototype HCoV-NL63 sequences isolated in The Netherlands in 1988 and 2003, indicating that these two subtypes may currently be cocirculating.

Human Infection with MERS coronavirus after exposure to infected camels, Saudi Arabia, 2013

NARCIS (Netherlands)

Memish, Ziad A.; Cotten, Matthew; Meyer, Benjamin; Watson, Simon J.; Alsahafi, Abdullah J.; Al Rabeeah, Abdullah A.; Corman, Victor Max; Sieberg, Andrea; Makhdoom, Hatem Q.; Assiri, Abdullah; Al Masri, Malaki; Aldabbagh, Souhaib; Bosch, Berend Jan|info:eu-repo/dai/nl/273306049; Beer, Martin; Müller, Marcel A.; Kellam, Paul; Drosten, Christian

2014-01-01

We investigated a case of human infection with Middle East respiratory syndrome coronavirus (MERS-CoV) after exposure to infected camels. Analysis of the whole human-derived virus and 15% of the camel-derived virus sequence yielded nucleotide polymorphism signatures suggestive of cross-species

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats

NARCIS (Netherlands)

Corman, Victor Max; Baldwin, Heather J.; Tateno, Adriana Fumie; Zerbinati, Rodrigo Melim; Annan, Augustina; Owusu, Michael; Nkrumah, Evans Ewald; Maganga, Gael Darren; Oppong, Samuel; Adu-Sarkodie, Yaw; Vallo, Peter; da Silva Filho, Luiz Vicente Ribeiro Ferreira; Leroy, Eric M.; Thiel, Volker; van der Hoek, Lia; Poon, Leo L. M.; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

2015-01-01

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related

Detection of potentially novel paramyxovirus and coronavirus viral RNA in bats and rats in the Mekong Delta region of southern Viet Nam.

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Berto, A; Anh, P H; Carrique-Mas, J J; Simmonds, P; Van Cuong, N; Tue, N T; Van Dung, N; Woolhouse, M E; Smith, I; Marsh, G A; Bryant, J E; Thwaites, G E; Baker, S; Rabaa, M A

2018-02-01

Bats and rodents are being increasingly recognized as reservoirs of emerging zoonotic viruses. Various studies have investigated bat viruses in tropical regions, but to date there are no data regarding viruses with zoonotic potential that circulate in bat and rat populations in Viet Nam. To address this paucity of data, we sampled three bat farms and three wet markets trading in rat meat in the Mekong Delta region of southern Viet Nam. Faecal and urine samples were screened for the presence of RNA from paramyxoviruses, coronaviruses and filoviruses. Paramyxovirus RNA was detected in 4 of 248 (1%) and 11 of 222 (4.9%) bat faecal and urine samples, respectively. Coronavirus RNA was detected in 55 of 248 (22%) of bat faecal samples; filovirus RNA was not detected in any of the bat samples. Further, coronavirus RNA was detected in 12 of 270 (4.4%) of rat faecal samples; all samples tested negative for paramyxovirus. Phylogenetic analysis revealed that the bat paramyxoviruses and bat and rat coronaviruses were related to viruses circulating in bat and rodent populations globally, but showed no cross-species mixing of viruses between bat and rat populations within Viet Nam. Our study shows that potentially novel variants of paramyxoviruses and coronaviruses commonly circulate in bat and rat populations in Viet Nam. Further characterization of the viruses and additional human and animal surveillance is required to evaluate the likelihood of viral spillover and to assess whether these viruses pose a risk to human health. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

Characterization of HCoV-229E fusion core: Implications for structure basis of coronavirus membrane fusion

International Nuclear Information System (INIS)

Liu Cheng; Feng Youjun; Gao Feng; Zhang Qiangmin; Wang Ming

2006-01-01

Human coronavirus 229E (HCoV-229E), a member of group I coronaviruses, has been identified as one of the major viral agents causing respiratory tract diseases in humans for nearly 40 years. However, the detailed molecular mechanism of the membrane fusion mediated by the spike (S) protein of HCoV-229E remains elusive. Here, we report, for the first time, a rationally designed fusion core of HCoV-229E (HR1-SGGRGG-HR2), which was in vitro produced in GST prokaryotic expression system. Multiple lines of experimental data including gel-filtration, chemical cross-linking, and circular diagram (CD) demonstrated that the HCoV-229E fusion core possesses the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle). 3D structure modeling presents its most-likely structure, similar to those of coronaviruses that have been well-documented. Collectively, HCoV-229E S protein belongs to the type I fusion protein, which is characterized by the existence of two heptad-repeat regions (HR1 and HR2), furthermore, the available knowledge concerning HCoV-229E fusion core may make it possible to design small molecule or polypeptide drugs targeting the membrane fusion, a crucial step of HCoV-229E infection

Sequence evidence for RNA recombination in field isolates of avian coronavirus infectious bronchitis virus

NARCIS (Netherlands)

Kusters, J G; Jager, E J; Niesters, H G; van der Zeijst, B A

1990-01-01

Under laboratory conditions coronaviruses were shown to have a high frequency of recombination. In The Netherlands, vaccination against infectious bronchitis virus (IBV) is performed with vaccines that contain several life-attenuated virus strains. These highly effective vaccines may create ideal

The Middle East respiratory syndrome coronavirus (MERS-CoV does not replicate in Syrian hamsters.

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Emmie de Wit

Full Text Available In 2012 a novel coronavirus, MERS-CoV, associated with severe respiratory disease emerged in the Arabian Peninsula. To date, 55 human cases have been reported, including 31 fatal cases. Several of the cases were likely a result of human-to-human transmission. The emergence of this novel coronavirus prompts the need for a small animal model to study the pathogenesis of this virus and to test the efficacy of potential intervention strategies. In this study we explored the use of Syrian hamsters as a small animal disease model, using intratracheal inoculation and inoculation via aerosol. Clinical signs of disease, virus replication, histological lesions, cytokine upregulation nor seroconversion were observed in any of the inoculated animals, indicating that MERS-CoV does not replicate in Syrian hamsters.

Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses

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Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M.; Weerasekara, Sahani; Hua, Duy H.; Kankanamalage, Anushka C. Galasiti; Groutas, William C.; Chang, Kyeong-Ok

2015-01-01

ABSTRACT Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats

The Paradox of Feline Coronavirus Pathogenesis: A Review

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Luciana Wanderley Myrrha

2011-01-01

Full Text Available Feline coronavirus (FCoV is an enveloped single-stranded RNA virus, of the family Coronaviridae and the order Nidovirales. FCoV is an important pathogen of wild and domestic cats and can cause a mild or apparently symptomless enteric infection, especially in kittens. FCoV is also associated with a lethal, systemic disease known as feline infectious peritonitis (FIP. Although the precise cause of FIP pathogenesis remains unclear, some hypotheses have been suggested. In this review we present results from different FCoV studies and attempt to elucidate existing theories on the pathogenesis of FCoV infection.

Clinical, hematological, and biochemical findings in puppies with coronavirus and parvovirus enteritis

Science.gov (United States)

Castro, Tatiana X.; Cubel Garcia, Rita de Cássia N.; Gonçalves, Luciana P. S.; Costa, Erika M.; Marcello, Gracy C.G.; Labarthe, Norma V.; Mendes-de-Almeida, Flavya

2013-01-01

The clinical and laboratory findings in puppies naturally infected with canine coronavirus (CCoV) and/or canine parvovirus (CPV) were compared with findings in uninfected puppies. Lymphopenia was the only parameter related to CCoV infection that was statistically significant; vomiting, anorexia, lethargy, hemorrhagic fluid diarrhea, leukopenia, lymphopenia, thrombocytopenia, hypoglycemia, and hypoproteinemia were correlated with CPV infection. PMID:24155496

SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum.

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Kèvin Knoops

2008-09-01

Full Text Available Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV, replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200-300 nm, and "vesicle packets" apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this "replication network" will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

International Nuclear Information System (INIS)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-01-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [ 3 H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating

Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

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Berkhout Ben

2006-12-01

Full Text Available Abstract Background The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU in Salisbury, UK. Results All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%. Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates carry a single ORF, 660 nt in size, encoding a single protein of 219 amino acids, which is a homologue of the ORF3 proteins encoded by HCoV-NL63 and PEDV. Conclusion Thus, the genome organization of the group 1b coronaviruses HCoV-NL63, PEDV and HCoV-229E is identical. It is possible that extensive culturing of the HCoV-229E laboratory strain resulted in truncation of ORF4. This may indicate that the protein is not essential in cell culture, but the highly conserved amino acid sequence of the ORF4 protein among clinical isolates suggests that the protein plays an important role in vivo.

Reassessment of the role of TSC, mTORC1 and microRNAs in amino acids-meditated translational control of TOP mRNAs.

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Ilona Patursky-Polischuk

Full Text Available TOP mRNAs encode components of the translational apparatus, and repression of their translation comprises one mechanism, by which cells encountering amino acid deprivation downregulate the biosynthesis of the protein synthesis machinery. This mode of regulation involves TSC as knockout of TSC1 or TSC2 rescued TOP mRNAs translation in amino acid-starved cells. The involvement of mTOR in translational control of TOP mRNAs is demonstrated by the ability of constitutively active mTOR to relieve the translational repression of TOP mRNA upon amino acid deprivation. Consistently, knockdown of this kinase as well as its inhibition by pharmacological means blocked amino acid-induced translational activation of these mRNAs. The signaling of amino acids to TOP mRNAs involves RagB, as overexpression of active RagB derepressed the translation of these mRNAs in amino acid-starved cells. Nonetheless, knockdown of raptor or rictor failed to suppress translational activation of TOP mRNAs by amino acids, suggesting that mTORC1 or mTORC2 plays a minor, if any, role in this mode of regulation. Finally, miR10a has previously been suggested to positively regulate the translation of TOP mRNAs. However, we show here that titration of this microRNA failed to downregulate the basal translation efficiency of TOP mRNAs. Moreover, Drosha knockdown or Dicer knockout, which carries out the first and second processing steps in microRNAs biosynthesis, respectively, failed to block the translational activation of TOP mRNAs by amino acid or serum stimulation. Evidently, these results are questioning the positive role of microRNAs in this mode of regulation.

Regional localization of suspensor mRNAs during early embryo development.

Science.gov (United States)

Weterings, K; Apuya, N R; Bi, Y; Fischer, R L; Harada, J J; Goldberg, R B

2001-11-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/beta-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote.

Regional Localization of Suspensor mRNAs during Early Embryo Development

Science.gov (United States)

Weterings, Koen; Apuya, Nestor R.; Bi, Yuping; Fischer, Robert L.; Harada, John J.; Goldberg, Robert B.

2001-01-01

We investigated gene activity within the giant embryos of the scarlet runner bean (Phaseolus coccineus) to gain understanding of the processes by which the apical and basal cells become specified to follow different developmental pathways after division of the zygote. We identified two mRNAs, designated G564 and C541, that accumulate specifically within the suspensor of globular-stage embryos. G564 mRNA accumulates uniformly throughout the suspensor, whereas C541 mRNA accumulates to a higher level within the large basal cells of the suspensor that anchor the embryo to the surrounding seed tissue. Both G564 and C541 mRNAs begin to accumulate shortly after fertilization and are present within the two basal cells of embryos at the four-cell stage. In contrast, at the same stage, these mRNAs are not detectable within the two descendants of the apical cell. Nor are they detectable within cells of the embryo sac before fertilization, including the egg cell. We used a G564/β-glucuronidase reporter gene to show that the G564 promoter is activated specifically within the basal region and suspensor of preglobular tobacco embryos. Analysis of the G564 promoter identified a sequence domain required for transcription within the suspensor that contains several copies of a conserved motif. These results show that derivatives of the apical and basal cells transcribe different genes as early as the four-cell stage of embryo development and suggest that the apical and basal cells are specified at the molecular level after division of the zygote. PMID:11701878

Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas).

Science.gov (United States)

Liu, Shengwang; Chen, Jianfei; Chen, Jinding; Kong, Xiangang; Shao, Yuhao; Han, Zongxi; Feng, Li; Cai, Xuehui; Gu, Shoulin; Liu, Ming

2005-03-01

Coronavirus-like viruses, designated peafowl/China/LKQ3/2003 (pf/CH/LKQ3/03) and teal/China/LDT3/2003 (tl/CH/LDT3/03), were isolated from a peafowl and a teal during virological surveillance in Guangdong province, China. Partial genomic sequence analysis showed that these isolates had the S-3-M-5-N gene order that is typical of avian coronaviruses. The spike, membrane and nucleocapsid protein genes of pf/CH/LKQ3/03 had >99 % identity to those of the avian infectious bronchitis coronavirus H120 vaccine strain (Massachusetts serotype) and other Massachusetts serotype isolates. Furthermore, when pf/CH/LKQ3/03 was inoculated experimentally into chickens (specific-pathogen-free), no disease signs were apparent. tl/CH/LDT3/03 had a spike protein gene with 95 % identity to that of a Chinese infectious bronchitis virus (IBV) isolate, although more extensive sequencing revealed the possibility that this strain may have undergone recombination. When inoculated into chickens, tl/CH/LDT3/03 resulted in the death of birds from nephritis. Taken together, this information suggests that pf/CH/LKQ3/03 might be a revertant, attenuated vaccine IBV strain, whereas tl/CH/LDT3/03 is a nephropathogenic field IBV strain, generated through recombination. The replication and non-pathogenic nature of IBV in domestic peafowl and teal under field conditions raises questions as to the role of these hosts as carriers of IBV and the potential that they may have to transmit virus to susceptible chicken populations.

Genome-wide analysis of codon usage bias in Bovine Coronavirus

OpenAIRE

Castells, Mat?as; Victoria, Mat?as; Colina, Rodney; Musto, H?ctor; Cristina, Juan

2017-01-01

Background Bovine coronavirus (BCoV) belong to the genus Betacoronavirus of the family Coronaviridae. BCoV are widespread around the world and cause enteric or respiratory infections among cattle, leading to important economic losses to the beef and dairy industry worldwide. To study the relation of codon usage among viruses and their hosts is essential to understand host-pathogen interaction, evasion from host?s immune system and evolution. Methods We performed a comprehensive analysis of co...

Association of seropositivity for influenza and coronaviruses with history of mood disorders and suicide attempts.

Science.gov (United States)

Okusaga, Olaoluwa; Yolken, Robert H; Langenberg, Patricia; Lapidus, Manana; Arling, Timothy A; Dickerson, Faith B; Scrandis, Debra A; Severance, Emily; Cabassa, Johanna A; Balis, Theodora; Postolache, Teodor T

2011-04-01

Anecdotal reports of mood disorder following infection with common respiratory viruses with neurotropic potential have been in existence since the last century. Nevertheless, systematic studies on the association between these viruses and mood disorders are lacking. Influenza A, B and coronavirus antibody titers were measured in 257 subjects with recurrent unipolar and bipolar disorder and healthy controls, by SCID. Pearson's χ² tests and logistic regression models were used to analyze associations between seropositivity for coronaviruses, influenza A and B viruses and the following: a) history of recurrent mood disorders b) having attempted suicide in the past c) uni- vs. bi-polarity and d) presence of psychotic symptoms during mood episodes. Seropositivity for influenza A (p=0.004), B (pmood disorders but not with the specific diagnosis of unipolar or bipolar depression. Seropositivity for influenza B was significantly associated with a history of suicide attempt (p=0.001) and history of psychotic symptoms (p=0.005). The design was cross-sectional. Socioeconomic factors, inflammatory markers, and axis II psychopathology were not assessed. The association of seropositivity for influenza and coronaviruses with a history of mood disorders, and influenza B with suicidal behavior require replication in larger longitudinal samples. The need for these studies is additionally supported by the high incidence of these viral infections, the high prevalence of mood disorders, and resilience of suicide epidemics. Copyright © 2010 Elsevier B.V. All rights reserved.

Molecular dynamics of Middle East Respiratory Syndrome Coronavirus (MERS CoV) fusion heptad repeat trimers

KAUST Repository

Kandeel, Mahmoud; Al-Taher, Abdulla; Li, Huifang; Schwingenschlö gl, Udo; Alnazawi, Mohamed

2018-01-01

Structural studies related to Middle East Respiratory Syndrome Coronavirus (MERS CoV) infection process are so limited. In this study, molecular dynamics (MD) simulation was carried out to unravel changes in the MERS CoV heptad repeat domains (HRs

Coronavirus envelope (E) protein remains at the site of assembly

International Nuclear Information System (INIS)

Venkatagopalan, Pavithra; Daskalova, Sasha M.; Lopez, Lisa A.; Dolezal, Kelly A.; Hogue, Brenda G.

2015-01-01

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes

Pathogenic characteristics of persistent feline enteric coronavirus infection in cats

Science.gov (United States)

Vogel, Liesbeth; Van der Lubben, Mariken; Te Lintelo, Eddie G.; Bekker, Cornelis P.J.; Geerts, Tamara; Schuijff, Leontine S.; Grinwis, Guy C.M.; Egberink, Herman F.; Rottier, Peter J.M.

2010-01-01

Feline coronaviruses (FCoV) comprise two biotypes: feline enteric coronaviruses (FECV) and feline infectious peritonitis viruses (FIPV). FECV is associated with asymptomatic persistent enteric infections, while FIPV causes feline infectious peritonitis (FIP), a usually fatal systemic disease in domestic cats and some wild Felidae. FIPV arises from FECV by mutation. FCoV also occur in two serotypes, I and II, of which the serotype I viruses are by far the most prevalent in the field. Yet, most of our knowledge about FCoV infections relates to serotype II viruses, particularly about the FIPV, mainly because type I viruses grow poorly in cell culture. Hence, the aim of the present work was the detailed study of the epidemiologically most relevant viruses, the avirulent serotype I viruses. Kittens were inoculated oronasally with different doses of two independent FECV field strains, UCD and RM. Persistent infection could be reproducibly established. The patterns of clinical symptoms, faecal virus shedding and seroconversion were monitored for up to 10 weeks revealing subtle but reproducible differences between the two viruses. Faecal virus, i.e. genomic RNA, was detected during persistent FECV infection only in the large intestine, downstream of the appendix, and could occasionally be observed also in the blood. The implications of our results, particularly our insights into the persistently infected state, are discussed. PMID:20663472

Coronavirus envelope (E) protein remains at the site of assembly

Energy Technology Data Exchange (ETDEWEB)

Venkatagopalan, Pavithra [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Daskalova, Sasha M. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); Department of Biochemistry and Chemistry, Arizona State University, Tempe, AZ 85287-5401 (United States); Lopez, Lisa A. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Molecular and Cellular Biology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Dolezal, Kelly A. [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States); Microbiology Graduate Program, Arizona State University, Tempe, AZ 85287-5401 (United States); Hogue, Brenda G., E-mail: Brenda.Hogue@asu.edu [The Biodesign Institute, Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ 85287-5401 (United States); School of Life Sciences, Arizona State University, Tempe, AZ 85287-5401 (United States)

2015-04-15

Coronaviruses (CoVs) assemble at endoplasmic reticulum Golgi intermediate compartment (ERGIC) membranes and egress from cells in cargo vesicles. Only a few molecules of the envelope (E) protein are assembled into virions. The role of E in morphogenesis is not fully understood. The cellular localization and dynamics of mouse hepatitis CoV A59 (MHV) E protein were investigated to further understanding of its role during infection. E protein localized in the ERGIC and Golgi with the amino and carboxy termini in the lumen and cytoplasm, respectively. E protein does not traffic to the cell surface. MHV was genetically engineered with a tetracysteine tag at the carboxy end of E. Fluorescence recovery after photobleaching (FRAP) showed that E is mobile in ERGIC/Golgi membranes. Correlative light electron microscopy (CLEM) confirmed the presence of E in Golgi cisternae. The results provide strong support that E proteins carry out their function(s) at the site of budding/assembly. - Highlights: • Mouse hepatitis coronavirus (MHV-CoV) E protein localizes in the ERGIC and Golgi. • MHV-CoV E does not transport to the cell surface. • MHV-CoV can be genetically engineered with a tetracysteine tag appended to E. • First FRAP and correlative light electron microscopy of a CoV E protein. • Live-cell imaging shows that E is mobile in ERGIC/Golgi membranes.

Middle East Respiratory Coronavirus Accessory Protein 4a Inhibits PKR-Mediated Antiviral Stress Responses

NARCIS (Netherlands)

Rabouw, Huib H; Langereis, Martijn A; Knaap, Robert C M; Dalebout, Tim J; Canton, Javier; Sola, Isabel; Enjuanes, Luis; Bredenbeek, Peter J; Kikkert, Marjolein; de Groot, Raoul J; van Kuppeveld, Frank J M

2016-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory infections that can be life-threatening. To establish an infection and spread, MERS-CoV, like most other viruses, must navigate through an intricate network of antiviral host responses. Besides the well-known type I

E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity

Energy Technology Data Exchange (ETDEWEB)

Vlasak, R.; Luytjes, W.; Leider, J.; Spaan, W.; Palese, P.

1988-12-01

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with (/sup 3/H)DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the esterase/receptor-destroying activity of BCV is associated with the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possible during virus entry or uncoating.

Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

Directory of Open Access Journals (Sweden)

Jazmina L G Cruz

2011-06-01

Full Text Available Transmissible gastroenteritis virus (TGEV genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7. Both the mutant and the parental (rTGEV-wt viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c, a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the

Biochemical and biophysical characterization of the transmissible gastroenteritis coronavirus fusion core

International Nuclear Information System (INIS)

Ma Guangpeng; Feng Youjun; Gao Feng; Wang Jinzi; Liu Cheng; Li Yijing

2005-01-01

Transmissible gastroenteritis coronavirus (TGEV) is one of the most destructive agents, responsible for the enteric infections that are lethal for suckling piglets, causing enormous economic loss to the porcine fostering industry every year. Although it has been known that TGEV spiker protein is essential for the viral entry for many years, the detail knowledge of the TGEV fusion protein core is still very limited. Here, we report that TGEV fusion core (HR1-SGGRGG-HR2), in vitro expressed in GST prokaryotic expression system, shares the typical properties of the trimer of coiled-coil heterodimer (six α-helix bundle), which has been confirmed by a combined series of biochemical and biophysical evidences including size exclusion chromatography (gel-filtration), chemical crossing, and circular diagram. The 3D homologous structure model presents its most likely structure, extremely similar to those of the coronaviruses documented. Taken together, TGEV spiker protein belongs to the class I fusion protein, characterized by the existence of two heptad-repeat (HR) regions, HR1 and HR2, and the present knowledge about the truncated TGEV fusion protein core may facilitate in the design of the small molecule or polypeptide drugs targeting the membrane fusion between TGEV and its host

The first complete genome sequences of clinical isolates of human coronavirus 229E

NARCIS (Netherlands)

Farsani, Seyed Mohammad Jazaeri; Dijkman, Ronald; Jebbink, Maarten F.; Goossens, Herman; Ieven, Margareta; Deijs, Martin; Molenkamp, Richard; van der Hoek, Lia

2012-01-01

Human coronavirus 229E has been identified in the mid-1960s, yet still only one full-genome sequence is available. This full-length sequence has been determined from the cDNA-clone Inf-1 that is based on the lab-adapted strain VR-740. Lab-adaptation might have resulted in genomic changes, due to

Immune evasion of porcine enteric coronaviruses and viral modulation of antiviral innate signaling.

Science.gov (United States)

Zhang, Qingzhan; Yoo, Dongwan

2016-12-02

Porcine epidemic diarrhea virus (PEDV) and porcine deltacoronavirus (PDCoV) are emerged and reemerging viruses in pigs, and together with transmissible gastroenteritis virus (TGEV), pose significant economic concerns to the swine industry. These viruses infect epithelial cells of the small intestine and cause watery diarrhea, dehydration, and a high mortality in neonatal piglets. Type I interferons (IFN-α/β) are major antiviral cytokines forming host innate immunity, and in turn, these enteric coronaviruses have evolved to modulate the host innate immune signaling during infection. Accumulating evidence however suggests that IFN induction and signaling in the intestinal epithelial cells differ from other epithelial cells, largely due to distinct features of the gut epithelial mucosal surface and commensal microflora, and it appears that type III interferon (IFN-λ) plays a key role to maintain the antiviral state in the gut. This review describes the recent understanding on the immune evasion strategies of porcine enteric coronaviruses and the role of different types of IFNs for intestinal antiviral innate immunity. Copyright © 2016 Elsevier B.V. All rights reserved.

Mutation of the S and 3c genes in genomes of feline coronaviruses.

Science.gov (United States)

Oguma, Keisuke; Ohno, Megumi; Yoshida, Mayuko; Sentsui, Hiroshi

2018-05-17

Feline coronavirus (FCoV) is classified into two biotypes based on its pathogenicity in cats: a feline enteric coronavirus of low pathogenicity and a highly virulent feline infectious peritonitis virus. It has been suspected that FCoV alters its biotype via mutations in the viral genome. The S and 3c genes of FCoV have been considered the candidates for viral pathogenicity conversion. In the present study, FCoVs were analyzed for the frequency and location of mutations in the S and 3c genes from faecal samples of cats in an animal shelter and the faeces, effusions, and tissues of cats that were referred to veterinary hospitals. Our results indicated that approximately 95% FCoVs in faeces did not carry mutations in the two genes. However, 80% FCoVs in effusion samples exhibited mutations in the S and 3c genes with remainder displaying a mutation in the S or 3c gene. It was also suggested that mutational analysis of the 3c gene could be useful for studying the horizontal transmission of FCoVs in multi-cat environments.

Design, synthesis and crystallographic analysis of nitrile-based broad-spectrum peptidomimetic inhibitors for coronavirus 3C-like proteases.

Science.gov (United States)

Chuck, Chi-Pang; Chen, Chao; Ke, Zhihai; Wan, David Chi-Cheong; Chow, Hak-Fun; Wong, Kam-Bo

2013-01-01

Coronaviral infection is associated with up to 5% of respiratory tract diseases. The 3C-like protease (3CL(pro)) of coronaviruses is required for proteolytic processing of polyproteins and viral replication, and is a promising target for the development of drugs against coronaviral infection. We designed and synthesized four nitrile-based peptidomimetic inhibitors with different N-terminal protective groups and different peptide length, and examined their inhibitory effect on the in-vitro enzymatic activity of 3CL(pro) of severe-acute-respiratory-syndrome-coronavirus. The IC(50) values of the inhibitors were in the range of 4.6-49 μM, demonstrating that the nitrile warhead can effectively inactivate the 3CL(pro) autocleavage process. The best inhibitor, Cbz-AVLQ-CN with an N-terminal carbobenzyloxy group, was ~10x more potent than the other inhibitors tested. Crystal structures of the enzyme-inhibitor complexes showed that the nitrile warhead inhibits 3CL(pro) by forming a covalent bond with the catalytic Cys145 residue, while the AVLQ peptide forms a number of favourable interactions with the S1-S4 substrate-binding pockets. We have further showed that the peptidomimetic inhibitor, Cbz-AVLQ-CN, has broad-spectrum inhibition against 3CL(pro) from human coronavirus strains 229E, NL63, OC43, HKU1, and infectious bronchitis virus, with IC(50) values ranging from 1.3 to 3.7 μM, but no detectable inhibition against caspase-3. In summary, we have shown that the nitrile-based peptidomimetic inhibitors are effective against 3CL(pro), and they inhibit 3CL(pro) from a broad range of coronaviruses. Our results provide further insights into the future design of drugs that could serve as a first line defence against coronaviral infection. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture.

OpenAIRE

Harrison , Sally; Tarpey , Ian; Rothwell , Lisa; Kasier , Pete; Hiscox , Julian

2007-01-01

Abstract The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry which, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross strain immunity. Effective antivira...

The Binding Sites of miR-619-5p in the mRNAs of Human and Orthologous Genes.

Science.gov (United States)

Atambayeva, Shara; Niyazova, Raigul; Ivashchenko, Anatoliy; Pyrkova, Anna; Pinsky, Ilya; Akimniyazova, Aigul; Labeit, Siegfried

2017-06-01

Normally, one miRNA interacts with the mRNA of one gene. However, there are miRNAs that can bind to many mRNAs, and one mRNA can be the target of many miRNAs. This significantly complicates the study of the properties of miRNAs and their diagnostic and medical applications. The search of 2,750 human microRNAs (miRNAs) binding sites in 12,175 mRNAs of human genes using the MirTarget program has been completed. For the binding sites of the miR-619-5p the hybridization free energy of the bonds was equal to 100% of the maximum potential free energy. The mRNAs of 201 human genes have complete complementary binding sites of miR-619-5p in the 3'UTR (214 sites), CDS (3 sites), and 5'UTR (4 sites). The mRNAs of CATAD1, ICA1L, GK5, POLH, and PRR11 genes have six miR-619-5p binding sites, and the mRNAs of OPA3 and CYP20A1 genes have eight and ten binding sites, respectively. All of these miR-619-5p binding sites are located in the 3'UTRs. The miR-619-5p binding site in the 5'UTR of mRNA of human USP29 gene is found in the mRNAs of orthologous genes of primates. Binding sites of miR-619-5p in the coding regions of mRNAs of C8H8orf44, C8orf44, and ISY1 genes encode the WLMPVIP oligopeptide, which is present in the orthologous proteins. Binding sites of miR-619-5p in the mRNAs of transcription factor genes ZNF429 and ZNF429 encode the AHACNP oligopeptide in another reading frame. Binding sites of miR-619-5p in the 3'UTRs of all human target genes are also present in the 3'UTRs of orthologous genes of mammals. The completely complementary binding sites for miR-619-5p are conservative in the orthologous mammalian genes. The majority of miR-619-5p binding sites are located in the 3'UTRs but some genes have miRNA binding sites in the 5'UTRs of mRNAs. Several genes have binding sites for miRNAs in the CDSs that are read in different open reading frames. Identical nucleotide sequences of binding sites encode different amino acids in different proteins. The binding sites of miR-619-5p

Automated extraction protocol for quantification of SARS-Coronavirus RNA in serum: an evaluation study

Directory of Open Access Journals (Sweden)

Lui Wing-bong

2006-02-01

Full Text Available Abstract Background We have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (SARS based on the detection of the SARS-coronavirus RNA in serum by real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR. In this study, we evaluated the feasibility of automating the serum RNA extraction procedure in order to increase the throughput of the assay. Methods An automated nucleic acid extraction platform using the MagNA Pure LC instrument (Roche Diagnostics was evaluated. We developed a modified protocol in compliance with the recommended biosafety guidelines from the World Health Organization based on the use of the MagNA Pure total nucleic acid large volume isolation kit for the extraction of SARS-coronavirus RNA. The modified protocol was compared with a column-based extraction kit (QIAamp viral RNA mini kit, Qiagen for quantitative performance, analytical sensitivity and precision. Results The newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the MagNA Pure LC instrument. However, the automated method was found to be less sensitive, less precise and led to consistently lower serum SARS-coronavirus concentrations when compared with the column-based extraction method. Conclusion As the diagnostic efficiency and prognostic value of the serum SARS-CoV RNA RT-PCR test is critically associated with the analytical sensitivity and quantitative performance contributed both by the RNA extraction and RT-PCR components of the test, we recommend the use of the column-based manual RNA extraction method.

Multiple Export Mechanisms for mRNAs

Science.gov (United States)

Delaleau, Mildred; Borden, Katherine L. B.

2015-01-01

Nuclear mRNA export plays an important role in gene expression. We describe the mechanisms of mRNA export including the importance of mRNP assembly, docking with the nuclear basket of the nuclear pore complex (NPC), transit through the central channel of the NPC and cytoplasmic release. We describe multiple mechanisms of mRNA export including NXF1 and CRM1 mediated pathways. Selective groups of mRNAs can be preferentially transported in order to respond to cellular stimuli. RNAs can be selected based on the presence of specific cis-acting RNA elements and binding of specific adaptor proteins. The role that dysregulation of this process plays in human disease is also discussed. PMID:26343730

Somatomedin-C/insulin-like growth factor-I and Insulin-like growth factor-II mRNAs in rate fetal and adult tissues

International Nuclear Information System (INIS)

Lund, P.K.; Moats-Staats, B.M.; Hynes, M.A.; Simmons, J.G.; Jansen, M.; D'ercole, A.J.; Van Wyk, J.J.

1986-01-01

Somatomedin-C or insulin-like growth factor I (Sm-C/IGF-I) and insulin-like growth factor II (IGF-II) have been implicated in the regulation of fetal growth and development. In the present study 32 P-labeled complementary DNA probes encoding human and mouse Sm-C/IGF-I and human IGF-II were used in Northern blot hybridizations to analyze rat Sm-C/IGF-I and IGF-II mRNAs in poly(A + ) RNAs from intestine, liver, lung, and brain of adult rats and fetal rats between day 14 and 17 of gestation. In fetal rats, all four tissues contained a major mRNA of 1.7 kilobase (kb) that hybridized with the human Sm-C/IGF-I cDNA and mRNAs of 7.5, 4.7, 1.7, and 1.2 kb that hybridized with the mouse Sm-C/IGF-I cDNA. Adult rat intestine, liver, and lung also contained these mRNAs but Sm-C/IGF-I mRNAs were not detected in adult rat brain. These findings provide direct support for prior observations that multiple tissues in the fetus synthesize immunoreactive Sm-C/IGF-I and imply a role for Sm-C/IGF-I in fetal development as well as postnatally. Multiple IGF-II mRNAs of estimated sizes 4.7, 3.9, 2.2, 1.75, and 1.2 kb were observed in fetal rat intestine, liver, lung, and brain. The 4.7- and 3.9-kb mRNAs were the major hybridizing IGF-II mRNAs in all fetal tissues. Higher abundance of IGF-II mRNAs in rat fetal tissues compared with adult tissues supports prior hypotheses, based on serum IGF-II concentrations, that IGF-II is predominantly a fetal somatomedin. IGF-II mRNAs are present, however, in some poly(A + ) RNAs from adult rat tissues. The brain was the only tissue in the adult rat where the 4.7- and 3.9-kb IGF-II mRNAs were consistently detected. These findings suggest that a role for IGF-II in the adult rat, particularly in the central nervous system, cannot be excluded

Multiple-targeted graphene-based nanocarrier for intracellular imaging of mRNAs

International Nuclear Information System (INIS)

Wang, Ying; Li, Zhaohui; Liu, Misha; Xu, Jinjin; Hu, Dehong; Lin, Yuehe; Li, Jinghong

2017-01-01

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labelled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis. - Graphical abstract: Schematic illustration of simultaneously multiple mRNAs monitoring inside single living breast cancer cell based on GO nanocarrier. In particular, the fluorescent signals could be monitored when Mn-SOD probe (red) and β-actin probe (green) hybridizes with their mRNA targets inside the living cells. Random probe (orange) was regarded as control probe for the sensing strategy. - Highlights: • A multiple-targeted GO nanocarrier was used for mRNAs imaging and expression changes after drug treatment can be monitored successfully. • Sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) m

Seroprevalence and risk factors for infection with equine coronavirus in healthy horses in the USA

NARCIS (Netherlands)

Kooijman, L.J.; James, K.; Mapes, S.M.; Theelen, M.J.P.; Pusterla, N.

2017-01-01

Equine coronavirus (ECoV) is considered an enteric pathogen of foals and has only recently been associated with infections in adult horses. Seroprevalence data is needed to better understand the epidemiology of ECoV in adult horses, evaluate diagnostic modalities and develop preventive measures. The

A reverse genetics system for avian coronavirus infectious bronchitis virus based on targeted RNA recombination

NARCIS (Netherlands)

van Beurden, Steven J; Berends, Alinda J; Krämer-Kühl, Annika; Spekreijse, Dieuwertje; Chénard, Gilles; Philipp, Hans-Christian; Mundt, Egbert; Rottier, Peter J M; Verheije, M Hélène

2017-01-01

BACKGROUND: Avian coronavirus infectious bronchitis virus (IBV) is a respiratory pathogen of chickens that causes severe economic losses in the poultry industry worldwide. Major advances in the study of the molecular biology of IBV have resulted from the development of reverse genetics systems for

The roles of transportation and transportation hubs in the propagation of influenza and coronaviruses: a systematic review.

Science.gov (United States)

Browne, Annie; Ahmad, Sacha St-Onge; Beck, Charles R; Nguyen-Van-Tam, Jonathan S

2016-01-01

Respiratory viruses spread in humans across wide geographical areas in short periods of time, resulting in high levels of morbidity and mortality. We undertook a systematic review to assess the evidence that air, ground and sea mass transportation systems or hubs are associated with propagating influenza and coronaviruses. Healthcare databases and sources of grey literature were searched using pre-defined criteria between April and June 2014. Two reviewers screened all identified records against the protocol, undertook risk of bias assessments and extracted data using a piloted form. Results were analysed using a narrative synthesis. Forty-one studies met the eligibility criteria. Risk of bias was high in the observational studies, moderate to high in the reviews and moderate to low in the modelling studies. In-flight influenza transmission was identified substantively on five flights with up to four confirmed and six suspected secondary cases per affected flight. Five studies highlighted the role of air travel in accelerating influenza spread to new areas. Influenza outbreaks aboard cruise ships affect 2-7% of passengers. Influenza transmission events have been observed aboard ground transport vehicles. High heterogeneity between studies and the inability to exclude other sources of infection means that the risk of influenza transmission from an index case to other passengers cannot be accurately quantified. A paucity of evidence was identified describing severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome coronavirus transmission events associated with transportation systems or hubs. Air transportation appears important in accelerating and amplifying influenza propagation. Transmission occurs aboard aeroplanes, at the destination and possibly at airports. Control measures to prevent influenza transmission on cruise ships are needed to reduce morbidity and mortality. There is no recent evidence of sea transport accelerating influenza

The persistent prevalence and evolution of cross-family recombinant coronavirus GCCDC1 among a bat population: a two-year follow-up.

Science.gov (United States)

Obameso, Joseph O; Li, Hong; Jia, Hao; Han, Min; Zhu, Shiyan; Huang, Canping; Zhao, Yuhui; Zhao, Min; Bai, Yu; Yuan, Fei; Zhao, Honglan; Peng, Xia; Xu, Wen; Tan, Wenjie; Zhao, Yingze; Yuen, Kwok-Yung; Liu, William J; Lu, Lin; Gao, George F

2017-12-01

Bats are connected with the increasing numbers of emerging and re-emerging viruses that may break the species barrier and spread into the human population. Coronaviruses are one of the most common viruses discovered in bats, which were considered as the natural source of recent human-susceptible coronaviruses, i.e. SARS-COV and MERS-CoV. Our previous study reported the discovery of a bat-derived putative cross-family recombinant coronavirus with a reovirus gene p10, named as Ro-BatCoV GCCDC1. In this report, through a two-year follow-up of a special bat population in one specific cave of south China, we illustrate that Ro-BatCoV GCCDC1 persistently circulates among bats. Notably, through the longitudinal observation, we identified the dynamic evolution of Ro-BatCoV GCCDC1 in bats represented by continuously recombination events. Our study provides the first glimpse of the virus evolution in one longitudinally observed bat population cohort and underlines the surveillance and pre-warning of potential interspecies transmittable viruses in bats.

Novel coronavirus and astrovirus in Delaware Bay shorebirds.

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Kirsi S Honkavuori

Full Text Available Wild birds are an important but to some extent under-studied reservoir for emerging pathogens. We used unbiased sequencing methods for virus discovery in shorebird samples from the Delaware Bay, USA; an important feeding ground for thousands of migratory birds.Analysis of shorebird fecal samples indicated the presence of a novel astrovirus and coronavirus. A sanderling sample yielded sequences with distant homology to avian nephritis virus 1, an astrovirus associated with acute nephritis in poultry. A ruddy turnstone sample yielded sequences with homology to deltacoronaviruses.Our findings highlight shorebirds as a virus reservoir and the need to closely monitor wild bird populations for the emergence of novel virus variants.

Synthesis and processing of structural and intracellular proteins of two enteric coronaviruses

International Nuclear Information System (INIS)

Sardinia, L.M.

1985-01-01

The synthesis and processing of virus-specific proteins of two economically important enteric coronaviruses, bovine enteric coronavirus (BCV) and transmissible gastroenteritis virus (TGEV), were studied at the molecular level. To determine the time of appearance of virus-specific proteins, virus-infected cells were labeled with 35 S-methionine at various times during infection, immunoprecipitated with specific hyperimmune ascitic fluid, and analyzed by SDS-polyacrylamide gel electrophoresis. The peak of BCV protein synthesis was found to be at 12 hours postinfection (hpi). The appearance of all virus-specific protein was coordinated. In contrast, the peak of TGEV protein synthesis was at 8 hpi, but the nucleocapsid proteins was present as early as 4 hpi. Virus-infected cells were treated with tunicamycin to ascertain the types of glycosidic linkages of the glycoproteins. The peplomer proteins of both viruses were sensitive to inhibition by tunicamycin indicating that they possessed N-linked carbohydrates. The matrix protein of TGEV was similarly affected. The matrix protein of BCV, however, was resistant to tunicamycin treatment and, therefore, has O-linked carbohydrates. Only the nucleocapsid protein of both viruses is phosphorylated as detected by radiolabeling with 32 P-orthophosphate. Pulse-chase studies and comparison of intracellular and virion proteins were done to detect precursor-product relationships

Novel coronaviruses, astroviruses, adenoviruses and circoviruses in insectivorous bats from northern China.

Science.gov (United States)

Han, H-J; Wen, H-L; Zhao, L; Liu, J-W; Luo, L-M; Zhou, C-M; Qin, X-R; Zhu, Y-L; Liu, M-M; Qi, R; Li, W-Q; Yu, H; Yu, X-J

2017-12-01

Bats are considered as the reservoirs of several emerging infectious disease, and novel viruses are continually found in bats all around the world. Studies conducted in southern China found that bats carried a variety of viruses. However, few studies have been conducted on bats in northern China, which harbours a diversity of endemic insectivorous bats. It is important to understand the prevalence and diversity of viruses circulating in bats in northern China. In this study, a total of 145 insectivorous bats representing six species were collected from northern China and screened with degenerate primers for viruses belonging to six families, including coronaviruses, astroviruses, hantaviruses, paramyxoviruses, adenoviruses and circoviruses. Our study found that four of the viruses screened for were positive and the overall detection rates for astroviruses, coronaviruses, adenoviruses and circoviruses in bats were 21.4%, 15.9%, 20% and 37.2%, respectively. In addition, we found that bats in northern China harboured a diversity of novel viruses. Common Serotine (Eptesicus serotinu), Fringed long-footed Myotis (Myotis fimriatus) and Peking Myotis (Myotis pequinius) were investigated in China for the first time. Our study provided new information on the ecology and phylogeny of bat-borne viruses. © 2017 The Authors. Zoonoses and Public Health Published by Blackwell Verlag GmbH.

In situ localization of phenylpropanoid biosynthetic mRNAs and proteins in Parsley (Petroselinum crispum)

International Nuclear Information System (INIS)

Reinold, S.; Hahlbrock, K.

1997-01-01

Using in situ RNA/RNA hybridization, enzyme immunolocalization, and histochemical techniques, several phenylpropanoid biosynthetic activities and products were localized in tissue sections from various aerial parts of parsley (Petroselinum crispum) plants at different developmental stages. The enzymes and corresponding mRNAs analyzed included two representatives of general phenylpropanoid metabolism: phenylalanine ammonia-lyase (PAL) and 4-coumarate: CoA ligase (4CL), and one representative each from two distinct branch pathways: chalcone synthase (CHS; flavonoids) and S-adenosyl-L-methionine: bergaptol O-methyltransferase (BMT; furanocoumarins). In almost all cases, the relative timing of accumulation differed greatly for mRNA and protein and indicated short expression periods and short half-lives for all mRNAs as compared to the proteins. PAL and 4CL occurred almost ubiquitously in cell type-specific patterns, and their mRNAs and proteins were always coordinately expressed, whereas the cell type-specific localization of flavonoid and furanocoumarin biosynthetic activities was to a large extent mutually exclusive. However, the distribution patterns of CHS and BMT, when superimposed, closely matched those of PAL and 4CL in nearly all tissues analysed, suggesting that the flavonoid and furanocoumarin pathways together constituted a large majority of the total phenylpropanoid biosynthetic activity. Differential sites of synthesis and accumulation indicating intercellular translocation were observed both for flavonoids and for furanocoumarins in oil ducts and the surrounding tissue. The widespread occurrence of both classes of compounds, as well as selected, pathway-specific mRNAs and enzymes, in many cell types of all parsley organs including various flower parts suggests additional functions beyond the previously established roles of flavonoids in UV protection and furanocoumarins in pathogen defence. (author)

LARP1 functions as a molecular switch for mTORC1-mediated translation of an essential class of mRNAs.

Science.gov (United States)

Hong, Sungki; Freeberg, Mallory A; Han, Ting; Kamath, Avani; Yao, Yao; Fukuda, Tomoko; Suzuki, Tsukasa; Kim, John K; Inoki, Ken

2017-06-26

The RNA binding protein, LARP1, has been proposed to function downstream of mTORC1 to regulate the translation of 5'TOP mRNAs such as those encoding ribosome proteins (RP). However, the roles of LARP1 in the translation of 5'TOP mRNAs are controversial and its regulatory roles in mTORC1-mediated translation remain unclear. Here we show that LARP1 is a direct substrate of mTORC1 and Akt/S6K1. Deep sequencing of LARP1-bound mRNAs reveal that non-phosphorylated LARP1 interacts with both 5' and 3'UTRs of RP mRNAs and inhibits their translation. Importantly, phosphorylation of LARP1 by mTORC1 and Akt/S6K1 dissociates it from 5'UTRs and relieves its inhibitory activity on RP mRNA translation. Concomitantly, phosphorylated LARP1 scaffolds mTORC1 on the 3'UTRs of translationally-competent RP mRNAs to facilitate mTORC1-dependent induction of translation initiation. Thus, in response to cellular mTOR activity, LARP1 serves as a phosphorylation-sensitive molecular switch for turning off or on RP mRNA translation and subsequent ribosome biogenesis.

European Surveillance for Pantropic Canine Coronavirus

Science.gov (United States)

Cordonnier, Nathalie; Demeter, Zoltan; Egberink, Herman; Elia, Gabriella; Grellet, Aurélien; Le Poder, Sophie; Mari, Viviana; Martella, Vito; Ntafis, Vasileios; von Reitzenstein, Marcela; Rottier, Peter J.; Rusvai, Miklos; Shields, Shelly; Xylouri, Eftychia; Xu, Zach; Buonavoglia, Canio

2013-01-01

Highly virulent pantropic canine coronavirus (CCoV) strains belonging to subtype IIa were recently identified in dogs. To assess the distribution of such strains in Europe, tissue samples were collected from 354 dogs that had died after displaying systemic disease in France (n = 92), Hungary (n = 75), Italy (n = 69), Greece (n = 87), The Netherlands (n = 27), Belgium (n = 4), and Bulgaria (n = 1). A total of 124 animals tested positive for CCoV, with 33 of them displaying the virus in extraintestinal tissues. Twenty-four CCoV strains (19.35% of the CCoV-positive dogs) detected in internal organs were characterized as subtype IIa and consequently assumed to be pantropic CCoVs. Sequence and phylogenetic analyses of the 5′ end of the spike protein gene showed that pantropic CCoV strains are closely related to each other, with the exception of two divergent French viruses that clustered with enteric strains. PMID:23100349

A Rare Case of Human Coronavirus 229E Associated with Acute Respiratory Distress Syndrome in a Healthy Adult

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Foula Vassilara

2018-01-01

Full Text Available Human coronavirus 229E (HCoV-229E is one of the first coronavirus strains being described. It is linked to common cold symptoms in healthy adults. Younger children and the elderly are considered vulnerable to developing lower respiratory tract infections (LRTIs. In particular, immunocompromised patients have been reported with severe and life-threatening LRTIs attributed to HCoV-229E. We report for the first time a case of LRTI and acute respiratory distress syndrome developed in a healthy adult with no comorbidities and HCoV-229E strain identified as the only causative agent. A 45-year-old female with a clear medical history presented with fever, cough, and headache. Respiratory tract infection was diagnosed, and empirical antibiotics were started. Within two days, she developed bilateral pleural effusions, diffuse consolidations, and ground glass opacities involving all lung fields. She needed immediate oxygen supply, while ABGs deteriorated and chest imaging and PaO2/FiO2 indicated ARDS. Early administration of systemic corticosteroids led to gradual clinical improvement. Multiplex PCR from nasal secretions was positive only for HCoV-229E and negative for multiple other pathogens. It remains to be elucidated how an immunocompetent adult developed a life-threatening LRTI caused by a “benign considered” coronavirus strain, the HCoV-229E.

Computational modeling of the bat HKU4 coronavirus 3CLpro inhibitors as a tool for the development of antivirals against the emerging Middle East respiratory syndrome (MERS) coronavirus.

Science.gov (United States)

Abuhammad, Areej; Al-Aqtash, Rua'a A; Anson, Brandon J; Mesecar, Andrew D; Taha, Mutasem O

2017-11-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that poses a major challenge to clinical management. The 3C-like protease (3CL pro ) is essential for viral replication and thus represents a potential target for antiviral drug development. Presently, very few data are available on MERS-CoV 3CL pro inhibition by small molecules. We conducted extensive exploration of the pharmacophoric space of a recently identified set of peptidomimetic inhibitors of the bat HKU4-CoV 3CL pro . HKU4-CoV 3CL pro shares high sequence identity (81%) with the MERS-CoV enzyme and thus represents a potential surrogate model for anti-MERS drug discovery. We used 2 well-established methods: Quantitative structure-activity relationship (QSAR)-guided modeling and docking-based comparative intermolecular contacts analysis. The established pharmacophore models highlight structural features needed for ligand recognition and revealed important binding-pocket regions involved in 3CL pro -ligand interactions. The best models were used as 3D queries to screen the National Cancer Institute database for novel nonpeptidomimetic 3CL pro inhibitors. The identified hits were tested for HKU4-CoV and MERS-CoV 3CL pro inhibition. Two hits, which share the phenylsulfonamide fragment, showed moderate inhibitory activity against the MERS-CoV 3CL pro and represent a potential starting point for the development of novel anti-MERS agents. To the best of our knowledge, this is the first pharmacophore modeling study supported by in vitro validation on the MERS-CoV 3CL pro . MERS-CoV is an emerging virus that is closely related to the bat HKU4-CoV. 3CL pro is a potential drug target for coronavirus infection. HKU4-CoV 3CL pro is a useful surrogate model for the identification of MERS-CoV 3CL pro enzyme inhibitors. dbCICA is a very robust modeling method for hit identification. The phenylsulfonamide scaffold represents a potential starting point for MERS coronavirus 3CL pro inhibitors

Microarray profiling and co-expression network analysis of circulating lncRNAs and mRNAs associated with major depressive disorder.

Directory of Open Access Journals (Sweden)

Zhifen Liu

Full Text Available LncRNAs, which represent one of the most highly expressed classes of ncRNAs in the brain, are becoming increasingly interesting with regard to brain functions and disorders. However, changes in the expression of regulatory lncRNAs in Major Depressive Disorder (MDD have not yet been reported. Using microarrays, we profiled the expression of 34834 lncRNAs and 39224 mRNAs in peripheral blood sampled from MDD patients as well as demographically-matched controls. Among these, we found that 2007 lncRNAs and 1667 mRNAs were differentially expressed, 17 of which were documented as depression-related gene in previous studies. Gene Ontology (GO and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to fundamental metabolic processes and neurodevelopment diseases. To investigate the potential regulatory roles of the differentially expressed lncRNAs on the mRNAs, we also constructed co-expression networks composed of the lncRNAs and mRNAs, which shows significant correlated patterns of expression. In the MDD-derived network, there were a greater number of nodes and connections than that in the control-derived network. The lncRNAs located at chr10:874695-874794, chr10:75873456-75873642, and chr3:47048304-47048512 may be important factors regulating the expression of mRNAs as they have previously been reported associations with MDD. This study is the first to explore genome-wide lncRNA expression and co-expression with mRNA patterns in MDD using microarray technology. We identified circulating lncRNAs that are aberrantly expressed in MDD and the results suggest that lncRNAs may contribute to the molecular pathogenesis of MDD.

Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

KAUST Repository

Guerreiro, Ana

2014-11-03

Background Following fertilization, the early proteomes of metazoans are defined by the translation of stored but repressed transcripts; further embryonic development relies on de novo transcription of the zygotic genome. During sexual development of Plasmodium berghei, a rodent model for human malaria species including P. falciparum, the stability of repressed mRNAs requires the translational repressors DOZI and CITH. When these repressors are absent, Plasmodium zygote development and transmission to the mosquito vector is halted, as hundreds of transcripts become destabilized. However, which mRNAs are direct targets of these RNA binding proteins, and thus subject to translational repression, is unknown. Results We identify the maternal mRNA contribution to post-fertilization development of P. berghei using RNA immunoprecipitation and microarray analysis. We find that 731 mRNAs, approximately 50% of the transcriptome, are associated with DOZI and CITH, allowing zygote development to proceed in the absence of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete crystalloid with an essential function for sporozoite development. Conclusions Our study details for the first time the P. berghei maternal repressome. This mRNA population provides the developing ookinete with coding potential for key molecules required for life-cycle progression, and that are likely to be critical for the transmission of the malaria parasite from the rodent and the human host to the mosquito vector.

Sequence features of E. coli mRNAs affect their degradation.

Directory of Open Access Journals (Sweden)

Gal Lenz

Full Text Available Degradation of mRNA in bacteria is a regulatory mechanism, providing an efficient way to fine-tune protein abundance in response to environmental changes. While the mechanisms responsible for initiation and subsequent propagation of mRNA degradation are well studied, the mRNA features that affect its stability are yet to be elucidated. We calculated three properties for each mRNA in the E. coli transcriptome: G+C content, tRNA adaptation index (tAI and folding energy. Each of these properties were then correlated with the experimental transcript half life measured for each transcript and detected significant correlations. A sliding window analysis identified the regions that displayed the maximal signal. The correlation between transcript half life and both G+C content and folding energy was strongest at the 5' termini of the mRNAs. Partial correlations showed that each of the parameters contributes separately to mRNA half life. Notably, mRNAs of recently-acquired genes in the E. coli genome, which have a distinct nucleotide composition, tend to be highly stable. This high stability may aid the evolutionary fixation of horizontally acquired genes.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

Science.gov (United States)

Guimaraes, Ana M S; Brandão, Paulo E; de Moraes, Wanderlei; Cubas, Zalmir S; Santos, Leonilda C; Villarreal, Laura Y B; Robes, Rogério R; Coelho, Fabiana M; Resende, Mauricio; Santos, Renata C F; Oliveira, Rosangela C; Yamaguti, Mauricio; Marques, Lucas M; Neto, Renata L; Buzinhani, Melissa; Marques, Regina; Messick, Joanne B; Biondo, Alexander W; Timenetsky, Jorge

2009-06-01

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

Genetic diversity of bats coronaviruses in the Atlantic Forest hotspot biome, Brazil.

Science.gov (United States)

Góes, Luiz Gustavo Bentim; Campos, Angélica Cristine de Almeida; Carvalho, Cristiano de; Ambar, Guilherme; Queiroz, Luzia Helena; Cruz-Neto, Ariovaldo Pereira; Munir, Muhammad; Durigon, Edison Luiz

2016-10-01

Bats are notorious reservoirs of genetically-diverse and high-profile pathogens, and are playing crucial roles in the emergence and re-emergence of viruses, both in human and in animals. In this report, we identified and characterized previously unknown and diverse genetic clusters of bat coronaviruses in the Atlantic Forest Biome, Brazil. These results highlight the virus richness of bats and their possible roles in the public health. Copyright © 2016 Elsevier B.V. All rights reserved.

Altered Pathogenesis of Porcine Respiratory Coronavirus in Pigs due to Immunosuppressive Effects of Dexamethasone: Implications for Corticosteroid Use in Treatment of Severe Acute Respiratory Syndrome Coronavirus▿

OpenAIRE

Jung, Kwonil; Alekseev, Konstantin P.; Zhang, Xinsheng; Cheon, Doo-Sung; Vlasova, Anastasia N.; Saif, Linda J.

2007-01-01

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] ...

Anchoring a Defined Sequence to the 55' Ends of mRNAs : The Bolt to Clone Rare Full Length mRNAs and Generate cDNA Libraries porn a Few Cells.

Science.gov (United States)

Baptiste, J; Milne Edwards, D; Delort, J; Mallet, J

1993-01-01

Among numerous applications, the polymerase chain reaction (PCR) (1,2) provides a convenient means to clone 5' ends of rare mRNAs and to generate cDNA libraries from tissue available in amounts too low to be processed by conventional methods. Basically, the amplification of cDNAs by the PCR requires the availability of the sequences of two stretches of the molecule to be amplified. A sequence can easily be imposed at the 5' end of the first-strand cDNAs (corresponding to the 3' end of the mRNAs) by priming the reverse transcription with a specific primer (for cloning the 5' end of rare messenger) or with an oligonucleotide tailored with a poly (dT) stretch (for cDNA library construction), taking advantage of the poly (A) sequence that is located at the 3' end of mRNAs. Several strategies have been devised to tag the 3' end of the ss-cDNAs (corresponding to the 55' end of the mRNAs). We (3) and others have described strategies based on the addition of a homopolymeric dG (4,5) or dA (6,7) tail using terminal deoxyribonucleotide transferase (TdT) ("anchor-PCR" [4]). However, this strategy has important limitations. The TdT reaction is difficult to control and has a low efficiency (unpublished observations). But most importantly, the return primers containing a homopolymeric (dC or dT) tail generate nonspecific amplifications, a phenomenon that prevents the isolation of low abundance mRNA species and/or interferes with the relative abundance of primary clones in the library. To circumvent these drawbacks, we have used two approaches. First, we devised a strategy based on a cRNA enrichment procedure, which has been useful to eliminate nonspecific-PCR products and to allow detection and cloning of cDNAs of low abundance (3). More recently, to avoid the nonspecific amplification resulting from the annealing of the homopolymeric tail oligonucleotide, we have developed a novel anchoring strategy that is based on the ligation of an oligonucleotide to the 35' end of ss

A mouse model for MERS coronavirus-induced acute respiratory distress syndrome.

Science.gov (United States)

Cockrell, Adam S; Yount, Boyd L; Scobey, Trevor; Jensen, Kara; Douglas, Madeline; Beall, Anne; Tang, Xian-Chun; Marasco, Wayne A; Heise, Mark T; Baric, Ralph S

2016-11-28

Middle East respiratory syndrome coronavirus (MERS-CoV) is a novel virus that emerged in 2012, causing acute respiratory distress syndrome (ARDS), severe pneumonia-like symptoms and multi-organ failure, with a case fatality rate of ∼36%. Limited clinical studies indicate that humans infected with MERS-CoV exhibit pathology consistent with the late stages of ARDS, which is reminiscent of the disease observed in patients infected with severe acute respiratory syndrome coronavirus. Models of MERS-CoV-induced severe respiratory disease have been difficult to achieve, and small-animal models traditionally used to investigate viral pathogenesis (mouse, hamster, guinea-pig and ferret) are naturally resistant to MERS-CoV. Therefore, we used CRISPR-Cas9 gene editing to modify the mouse genome to encode two amino acids (positions 288 and 330) that match the human sequence in the dipeptidyl peptidase 4 receptor, making mice susceptible to MERS-CoV infection and replication. Serial MERS-CoV passage in these engineered mice was then used to generate a mouse-adapted virus that replicated efficiently within the lungs and evoked symptoms indicative of severe ARDS, including decreased survival, extreme weight loss, decreased pulmonary function, pulmonary haemorrhage and pathological signs indicative of end-stage lung disease. Importantly, therapeutic countermeasures comprising MERS-CoV neutralizing antibody treatment or a MERS-CoV spike protein vaccine protected the engineered mice against MERS-CoV-induced ARDS.

Regulation of RNA-binding proteins affinity to export receptors enables the nuclear basket proteins to distinguish and retain aberrant mRNAs.

Science.gov (United States)

Soheilypour, M; Mofrad, M R K

2016-11-02

Export of messenger ribonucleic acids (mRNAs) into the cytoplasm is a fundamental step in gene regulation processes, which is meticulously quality controlled by highly efficient mechanisms in eukaryotic cells. Yet, it remains unclear how the aberrant mRNAs are recognized and retained inside the nucleus. Using a new modelling approach for complex systems, namely the agent-based modelling (ABM) approach, we develop a minimal model of the mRNA quality control (QC) mechanism. Our results demonstrate that regulation of the affinity of RNA-binding proteins (RBPs) to export receptors along with the weak interaction between the nuclear basket protein (Mlp1 or Tpr) and RBPs are the minimum requirements to distinguish and retain aberrant mRNAs. Our results show that the affinity between Tpr and RBPs is optimized to maximize the retention of aberrant mRNAs. In addition, we demonstrate how the length of mRNA affects the QC process. Since longer mRNAs spend more time in the nuclear basket to form a compact conformation and initiate their export, nuclear basket proteins could more easily capture and retain them inside the nucleus.

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein

NARCIS (Netherlands)

Godeke, G J; de Haan, Cornelis A M; Rossen, J W; Vennema, H; Rottier, P J

The type I glycoprotein S of coronavirus, trimers of which constitute the typical viral spikes, is assembled into virions through noncovalent interactions with the M protein. Here we demonstrate that incorporation is mediated by the short carboxy-terminal segment comprising the transmembrane and

Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

DEFF Research Database (Denmark)

Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne

2003-01-01

site of transcription, and known S. cerevisiae nuclear structures such as the nucleolus and the nucleolar body. Our results show that retained SSA4 RNA localizes to an area in close proximity to the SSA4 locus. On deletion of Rrp6p and release from the genomic locus, heat shock mRNAs produced...

Heterogeneous Ribosomes Preferentially Translate Distinct Subpools of mRNAs Genome-wide.

Science.gov (United States)

Shi, Zhen; Fujii, Kotaro; Kovary, Kyle M; Genuth, Naomi R; Röst, Hannes L; Teruel, Mary N; Barna, Maria

2017-07-06

Emerging studies have linked the ribosome to more selective control of gene regulation. However, an outstanding question is whether ribosome heterogeneity at the level of core ribosomal proteins (RPs) exists and enables ribosomes to preferentially translate specific mRNAs genome-wide. Here, we measured the absolute abundance of RPs in translating ribosomes and profiled transcripts that are enriched or depleted from select subsets of ribosomes within embryonic stem cells. We find that heterogeneity in RP composition endows ribosomes with differential selectivity for translating subpools of transcripts, including those controlling metabolism, cell cycle, and development. As an example, mRNAs enriched in binding to RPL10A/uL1-containing ribosomes are shown to require RPL10A/uL1 for their efficient translation. Within several of these transcripts, this level of regulation is mediated, at least in part, by internal ribosome entry sites. Together, these results reveal a critical functional link between ribosome heterogeneity and the post-transcriptional circuitry of gene expression. Copyright © 2017 Elsevier Inc. All rights reserved.

Establishment of feline intestinal epithelial cell cultures for the propagation and study of feline enteric coronaviruses

Science.gov (United States)

2013-01-01

Feline infectious peritonitis (FIP) is the most feared infectious cause of death in cats, induced by feline infectious peritonitis virus (FIPV). This coronavirus is a virulent mutant of the harmless, ubiquitous feline enteric coronavirus (FECV). To date, feline coronavirus (FCoV) research has been hampered by the lack of susceptible cell lines for the propagation of serotype I FCoVs. In this study, long-term feline intestinal epithelial cell cultures were established from primary ileocytes and colonocytes by simian virus 40 (SV40) T-antigen- and human Telomerase Reverse Transcriptase (hTERT)-induced immortalization. Subsequently, these cultures were evaluated for their usability in FCoV research. Firstly, the replication capacity of the serotype II strains WSU 79–1683 and WSU 79–1146 was studied in the continuous cultures as was done for the primary cultures. In accordance with the results obtained in primary cultures, FCoV WSU 79–1683 still replicated significantly more efficient compared to FCoV WSU 79–1146 in both continuous cultures. In addition, the cultures were inoculated with faecal suspensions from healthy cats and with faecal or tissue suspensions from FIP cats. The cultures were susceptible to infection with different serotype I enteric strains and two of these strains were further propagated. No infection was seen in cultures inoculated with FIPV tissue homogenates. In conclusion, a new reliable model for FCoV investigation and growth of enteric field strains was established. In contrast to FIPV strains, FECVs showed a clear tropism for intestinal epithelial cells, giving an explanation for the observation that FECV is the main pathotype circulating among cats. PMID:23964891

Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia : a nationwide, cross-sectional, serological study

NARCIS (Netherlands)

Müller, Marcel A; Meyer, Benjamin; Corman, Victor M; Al-Masri, Malak; Turkestani, Abdulhafeez; Ritz, Daniel; Sieberg, Andrea; Aldabbagh, Souhaib; Bosch, Berend-J; Lattwein, Erik; Alhakeem, Raafat F; Assiri, Abdullah M; Albarrak, Ali M; Al-Shangiti, Ali M; Al-Tawfiq, Jaffar A; Wikramaratna, Paul; Alrabeeah, Abdullah A; Drosten, Christian; Memish, Ziad A

2015-01-01

BACKGROUND: Scientific evidence suggests that dromedary camels are the intermediary host for the Middle East respiratory syndrome coronavirus (MERS-CoV). However, the actual number of infections in people who have had contact with camels is unknown and most index patients cannot recall any such

Genome-wide analysis of uncapped mRNAs under heat stress in Arabidopsis

Directory of Open Access Journals (Sweden)

Emilio Gutierrez-Beltran

2015-09-01

Full Text Available Recently, we have showed that Tudor Staphylococcal Nuclease (TSN or Tudor-SN proteins (TSN1 and TSN2 are localized in cytoplasmic messenger ribonucleoprotein (mRNP complexes called stress granules (SG and processing bodies (PB under heat stress in Arabidopsis. One of the primary functions of these mRNP complexes is mRNA decay, which generates uncapped mRNAs by the action of endonucleases and decapping enzymes (Thomas et al., 2011 [1]. In order to figure out whether TSN proteins could be implicated in mRNA decay, we isolated uncapped and total mRNAs of Wild type (WT; Col and Ler and TSN double knock-out (tsn1tsn2 seedlings grown under heat stress (39 °C for 40 min and control (23 °C conditions. Here, we provide the experimental procedure to reproduce the results (NCBI GEO accession number GSE63522 published by Gutierrez-Beltran et al. (2015 in The Plant Cell [2].

SARS-coronavirus spike S2 domain flanked by cysteine residues C822 and C833 is important for activation of membrane fusion

International Nuclear Information System (INIS)

Madu, Ikenna G.; Belouzard, Sandrine; Whittaker, Gary R.

2009-01-01

The S2 domain of the coronavirus spike (S) protein is known to be responsible for mediating membrane fusion. In addition to a well-recognized cleavage site at the S1-S2 boundary, a second proteolytic cleavage site has been identified in the severe acute respiratory syndrome coronavirus (SARS-CoV) S2 domain (R797). C-terminal to this S2 cleavage site is a conserved region flanked by cysteine residues C822 and C833. Here, we investigated the importance of this well conserved region for SARS-CoV S-mediated fusion activation. We show that the residues between C822-C833 are well conserved across all coronaviruses. Mutagenic analysis of SARS-CoV S, combined with cell-cell fusion and pseudotyped virion infectivity assays, showed a critical role for the core-conserved residues C822, D830, L831, and C833. Based on available predictive models, we propose that the conserved domain flanked by cysteines 822 and 833 forms a loop structure that interacts with components of the SARS-CoV S trimer to control the activation of membrane fusion.

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

OpenAIRE

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. Th...

Characterization of the expression and immunogenicity of the ns4b protein of human coronavirus 229E

DEFF Research Database (Denmark)

Chagnon, F; Lamarre, A; Lachance, C

1998-01-01

to demonstrate the expression of ns4b in HCV-229E-infected cells using flow cytometry. Given a previously reported contiguous five amino acid shared region between ns4b and myelin basic protein, a purified recombinant histidine-tagged ns4b protein and (or) human myelin basic protein were injected into mice......Sequencing of complementary DNAs prepared from various coronaviruses has revealed open reading frames encoding putative proteins that are yet to be characterized and are so far only described as nonstructural (ns). As a first step in the elucidation of its function, we characterized the expression...... and immunogenicity of the ns4b gene product from strain 229E of human coronavirus (HCV-229E), a respiratory virus with a neurotropic potential. The gene was cloned and expressed in bacteria. A fusion protein of ns4b with maltose-binding protein was injected into rabbits to generate specific antibodies that were used...

Roles of viral and cellular proteins in the expression of alternatively spliced HTLV-1 pX mRNAs

International Nuclear Information System (INIS)

Princler, Gerald L.; Julias, John G.; Hughes, Stephen H.; Derse, David

2003-01-01

The human T cell leukemia virus type 1 (HTLV-1) genome contains a cluster of at least five open reading frames (ORFs) near the 3' terminus within the pX region. The pX ORFs are encoded by mono- or bicistronic mRNAs that are generated by alternative splicing. The various pX mRNAs result from skipping of the internal exon (2-exon versus 3-exon isofoms) or from the utilization of alternative splice acceptor sites in the terminal exon. The Rex and Tax proteins, encoded by ORFs X-III and X-IV, have been studied intensively and are encoded by the most abundant of the alternative 3-exon mRNAs. The protein products of the other pX ORFs have not been detected in HTLV-1-infected cell lines and the levels of the corresponding mRNAs have not been accurately established. We have used real-time RT-PCR with splice-site specific primers to accurately measure the levels of individual pX mRNA species in chronically infected T cell lines. We have asked whether virus regulatory proteins or ectopic expression of cellular factors influence pX mRNA splicing in cells that were transfected with HTLV-1 provirus clones. In chronically infected cell lines, the pX-tax/rex mRNA was present at 500- to 2500-fold higher levels than the pX-tax-orfII mRNA and at approximately 1000-fold higher levels than pX-rex-orfI mRNA. Chronically infected cell lines that contain numerous defective proviruses expressed 2-exon forms of pX mRNAs at significantly higher levels compared to cell lines that contain a single full-length provirus. Cells transfected with provirus expression plasmids expressed similar relative amounts of 3-exon pX mRNAs but lower levels of 2-exon mRNA forms compared to cells containing a single, full-length provirus. The pX mRNA expression patterns were nearly identical in cells transfected with wild-type, Tax-minus, or Rex-minus proviruses. Cotransfection of cells with HTLV-1 provirus in combination with SF2/ASF expression plasmid resulted in a relative increase in pX-tax/rex m

Differential expression of the MERS-coronavirus receptor in the upper respiratory tract of humans and dromedary camels

NARCIS (Netherlands)

Widagdo, W; Raj, V Stalin; Schipper, Debby; Kolijn, Kimberley; van Leenders, Geert J L H; Bosch, Berend J; Bensaid, Albert; Segalés, Joaquim; Baumgärtner, Wolfgang; Osterhaus, Albert D M E; Koopmans, Marion P; van den Brand, Judith M A; Haagmans, Bart L

Middle East respiratory syndrome coronavirus (MERS-CoV) is not efficiently transmitted between humans, but it is highly prevalent in dromedary camels. Here we report that the MERS-CoV receptor - dipeptidyl peptidase 4 (DPP4) - is expressed in the upper respiratory tract epithelium of camels but not

Genomic Analysis of 15 Human Coronaviruses OC43 (HCoV-OC43s Circulating in France from 2001 to 2013 Reveals a High Intra-Specific Diversity with New Recombinant Genotypes

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Nathalie Kin

2015-05-01

Full Text Available Human coronavirus OC43 (HCoV-OC43 is one of five currently circulating human coronaviruses responsible for respiratory infections. Like all coronaviruses, it is characterized by its genome’s high plasticity. The objectives of the current study were to detect genetically distinct genotypes and eventually recombinant genotypes in samples collected in Lower Normandy between 2001 and 2013. To this end, we sequenced complete nsp12, S, and N genes of 15 molecular isolates of HCoV-OC43 from clinical samples and compared them to available data from the USA, Belgium, and Hong-Kong. A new cluster E was invariably detected from nsp12, S, and N data while the analysis of nsp12 and N genes revealed the existence of new F and G clusters respectively. The association of these different clusters of genes in our specimens led to the description of thirteen genetically distinct genotypes, among which eight recombinant viruses were discovered. Identification of these recombinant viruses, together with temporal analysis and tMRCA estimation, provides important information for understanding the dynamics of the evolution of these epidemic coronaviruses.

Central ions and lateral asparagine/glutamine zippers stabilize the post-fusion hairpin conformation of the SARS coronavirus spike glycoprotein

International Nuclear Information System (INIS)

Duquerroy, Stephane; Vigouroux, Armelle; Rottier, Peter J.M.; Rey, Felix A.; Jan Bosch, Berend

2005-01-01

The coronavirus spike glycoprotein is a class I membrane fusion protein with two characteristic heptad repeat regions (HR1 and HR2) in its ectodomain. Here, we report the X-ray structure of a previously characterized HR1/HR2 complex of the severe acute respiratory syndrome coronavirus spike protein. As expected, the HR1 and HR2 segments are organized in antiparallel orientations within a rod-like molecule. The HR1 helices form an exceptionally long (120 A) internal coiled coil stabilized by hydrophobic and polar interactions. A striking arrangement of conserved asparagine and glutamine residues of HR1 propagates from two central chloride ions, providing hydrogen-bonding 'zippers' that strongly constrain the path of the HR2 main chain, forcing it to adopt an extended conformation at either end of a short HR2 α-helix

Human coronavirus OC43 causes influenza-like illness in residents and staff of aged-care facilities in Melbourne, Australia.

Science.gov (United States)

Birch, C. J.; Clothier, H. J.; Seccull, A.; Tran, T.; Catton, M. C.; Lambert, S. B.; Druce, J. D.

2005-01-01

Three outbreaks of respiratory illness associated with human coronavirus HCoV-OC43 infection occurred in geographically unrelated aged-care facilities in Melbourne, Australia during August and September 2002. On clinical and epidemiological grounds the outbreaks were first thought to be caused by influenza virus. HCoV-OC43 was detected by RT-PCR in 16 out of 27 (59%) specimens and was the only virus detected at the time of sampling. Common clinical manifestations were cough (74%), rhinorrhoea (59%) and sore throat (53%). Attack rates and symptoms were similar in residents and staff across the facilities. HCoV-OC43 was also detected in surveillance and diagnostic respiratory samples in the same months. These outbreaks establish this virus as a cause of morbidity in aged-care facilities and add to increasing evidence of the significance of coronavirus infections. PMID:15816152

Middle east respiratory syndrome coronavirus spike protein delivered by modified vaccinia virus ankara efficiently induces virus-neutralizing antibodies

NARCIS (Netherlands)

F. Song (Fei); R. Fux (Robert); L.B.V. Provacia (Lisette); A. Volz (Asisa); M. Eickmann; S. Becker (Stephan); A.D.M.E. Osterhaus (Albert); B.L. Haagmans (Bart); G. Suttera (Gerd)

2013-01-01

textabstractMiddle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic

Biodiversity impact of host interferon-stimulated-gene-product 15 on the coronavirus Papain-like protease deISGylase functions

Science.gov (United States)

Coronaviruses are single-stranded, positive sense RNA viruses whose members have severe impact on human health and cause significant economic hardships. Some pertinent examples include severe acute and Middle East respiratory syndromes (SARS-CoV; MERS-CoV), porcine epidemic diarrhea virus (PEDV), an...

Bovine coronavirus in naturally andexperimentally exposed calves; viralshedding and the potential for transmission

OpenAIRE

Oma, Veslemøy Sunniva; Tråven, Madeleine; Alenius, S.; Myrmel, Mette; Stokstad, Maria

2016-01-01

Background Bovine coronavirus (BCoV) is a widely distributed pathogen, causing disease and economic losses in the cattle industry worldwide. Prevention of virus spread is impeded by a lack of basic knowledge concerning viral shedding and transmission potential in individual animals. The aims of the study were to investigate the duration and quantity of BCoV shedding in feces and nasal secretions related to clinical signs, the presence of virus in blood and tissues and to test the hypothesis t...

Evaluation of bovine coronavirus antibody levels, virus shedding, and respiratory disease incidence throughout the beef cattle production cycle

Science.gov (United States)

Objective- Determine how levels of serum antibody to bovine coronavirus (BCV) are related to virus shedding patterns and respiratory disease incidence in beef calves at various production stages. Animals- 890 crossbred beef calves from four separately managed herds at the U.S. Meat Animal Research C...

Death of a dogma: eukaryotic mRNAs can code for more than one protein.

Science.gov (United States)

Mouilleron, Hélène; Delcourt, Vivian; Roucou, Xavier

2016-01-08

mRNAs carry the genetic information that is translated by ribosomes. The traditional view of a mature eukaryotic mRNA is a molecule with three main regions, the 5' UTR, the protein coding open reading frame (ORF) or coding sequence (CDS), and the 3' UTR. This concept assumes that ribosomes translate one ORF only, generally the longest one, and produce one protein. As a result, in the early days of genomics and bioinformatics, one CDS was associated with each protein-coding gene. This fundamental concept of a single CDS is being challenged by increasing experimental evidence indicating that annotated proteins are not the only proteins translated from mRNAs. In particular, mass spectrometry (MS)-based proteomics and ribosome profiling have detected productive translation of alternative open reading frames. In several cases, the alternative and annotated proteins interact. Thus, the expression of two or more proteins translated from the same mRNA may offer a mechanism to ensure the co-expression of proteins which have functional interactions. Translational mechanisms already described in eukaryotic cells indicate that the cellular machinery is able to translate different CDSs from a single viral or cellular mRNA. In addition to summarizing data showing that the protein coding potential of eukaryotic mRNAs has been underestimated, this review aims to challenge the single translated CDS dogma. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

TH and DCX mRNAs in peripheral blood and bone marrow predict outcome in metastatic neuroblastoma patients.

Science.gov (United States)

Yáñez, Yania; Hervás, David; Grau, Elena; Oltra, Silvestre; Pérez, Gema; Palanca, Sarai; Bermúdez, Mar; Márquez, Catalina; Cañete, Adela; Castel, Victoria

2016-03-01

In metastatic neuroblastoma (NB) patients, accurate risk stratification and disease monitoring would reduce relapse probabilities. This study aims to evaluate the independent prognostic significance of detecting tyrosine hydroxylase (TH) and doublecortin (DCX) mRNAs by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in peripheral blood (PB) and bone marrow (BM) samples from metastatic NB patients. RT-qPCR was performed on PB and BM samples from metastatic NB patients at diagnosis, post-induction therapy and at the end of treatment for TH and DCX mRNAs detection. High levels of TH and DCX mRNAs when detected in PB and BM at diagnosis independently predicted worse outcome in a cohort of 162 metastatic NB. In the subgroup of high-risk metastatic NB, TH mRNA detected in PB remained as independent predictor of EFS and OS at diagnosis. After the induction therapy, high levels of TH mRNA in PB and DCX mRNA in BM independently predicted poor EFS and OS. Furthermore TH mRNA when detected in BM predicted worse EFS. TH mRNA in PB samples at the end of treatment is an independent predictor of worse outcome. TH and DCX mRNAs levels in PB and BM assessed by RT-qPCR should be considered in new pre-treatment risk stratification strategies to reliable estimate outcome differences in metastatic NB patients. In those high-risk metastatic NB, TH and DCX mRNA quantification could be used for the assessment of response to treatment and for early detection of progressive disease or relapses.

Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.

Science.gov (United States)

Mohamed, J Shaik; Benninghoff, Abby D; Holt, G Joan; Khan, Izhar A

2007-02-01

The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

Infection of cats with atypical feline coronaviruses harbouring a truncated form of the canine type I non-structural ORF3 gene.

Science.gov (United States)

Le Poder, Sophie; Pham-Hung d'Alexandry d'Orangiani, Anne-Laure; Duarte, Lidia; Fournier, Annie; Horhogea, Cristina; Pinhas, Carine; Vabret, Astrid; Eloit, Marc

2013-12-01

Feline and canine coronaviruses (FCoV and CCoV, respectively) are common pathogens of cats and dogs sometimes leading to lethal infections named feline infectious peritonitis (FIP) and canine pantropic coronavirus infection. FCoV and CCoV are each subdivided into two serotypes, FCoV-I/II and CCoV-I/II. A phylogenetic relationship is evident between, on one hand, CCoV-I/FCoV-I, and on the other hand, CCoV-II/FCoV-II, suggesting that interspecies transmission can occur. The aim of the present study was to evaluate the prevalence of coronavirus (CoV)-infected cats according to their contact with dogs and to genetically analyse the CoV strains infecting cats. From 2003 to 2009, we collected 88 faecal samples from healthy cats and 11 ascitic fluids from FIP cats. We investigated the possible contact with dog in the household and collected dogs samples if appropriate. Out of 99 cat samples, 26 were coronavirus positive, with six cats living with at least one dog, thus showing that contact with dogs does not appear as a predisposing factor for cats CoV infections. Molecular and phylogenetic analyses of FCoV strains were conducted using partial N and S sequences. Six divergent strains were identified with the N gene clustering with CCoV-I whereas the 3' end of S was related to FCoV-I. Further analysis on those six samples was attempted by researching the presence of the ORF3 gene, the latter being peculiar to CCoV-I to date. We succeeded to amplify the ORF3 gene in five samples out of six. Thus, our data strongly suggest the circulation of atypical FCoV strains harbouring the CCoV-I ORF3 gene among cats. Moreover, the ORF3 genes recovered from the feline strains exhibited shared deletions, never described before, suggesting that these deletions could be critical in the adaptation of these strains to the feline host. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Proteolytic activation of the SARS-coronavirus spike protein: cutting enzymes at the cutting edge of antiviral research.

Science.gov (United States)

Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan

2013-12-01

The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.

The coronavirus transmissible gastroenteritis virus causes infection after receptor-mediated endocytosis and acid-dependent fusion with an intracellular compartment

DEFF Research Database (Denmark)

Hansen, Gert Helge; Delmas, B; Besnardeau, L

1998-01-01

Aminopeptidase N is a species-specific receptor for transmissible gastroenteritis virus (TGEV), which infects piglets, and for the 229E virus, which infects humans. It is not known whether these coronaviruses are endocytosed before fusion with a membrane of the target cell, causing a productive...

Multiple-Targeted Graphene-based Nanocarrier for Intracellular Imaging of mRNAs

Energy Technology Data Exchange (ETDEWEB)

Wang, Ying; Li, Zhaohui; Liu, Misha; Hu, Dehong; Lin, Yuehe; Li, Jinghong

2017-08-29

Simultaneous detection and imaging of multiple intracellular messenger RNA (mRNAs) hold great significant for early cancer diagnostics and preventive medicine development. Herein, we propose a multiple-targeted graphene oxide (GO) nanocarrier that can simultaneously detect and image different type mRNAs in living cells. First of all, in vitro detection of multiple targets have been realized successfully based on the multiple-targeted GO nanocarrier with linear relationship ranging from 3 nM to 200 nM, as well as sensitive detection limit of 1.84 nM for manganese superoxide dismutase (Mn-SOD) mRNA and 2.45 nM for β-actin mRNA. Additionally, this nanosensing platform composed of fluorescent labeled single strand DNA probes and GO nanocarrier can identify Mn-SOD mRNA and endogenous mRNA of β-actin in living cancer cells, showing rapid response, high specificity, nuclease stability, and good biocompatibility during the cell imaging. Thirdly, changes of the expression levels of mRNA in living cells before or after the drug treatment can be monitored successfully. By using multiple ssDNA as probes and GO nanocarrier as the cellular delivery cargo, the proposed simultaneous multiple-targeted sensing platform will be of great potential as a powerful tool for intracellular trafficking process from basic research to clinical diagnosis.

Expression of the somatostatin receptor family mRNAs in lung cancer

International Nuclear Information System (INIS)

Wang Jing; Wang Liangang; Deng Jinglan; Wu Shengxi

2000-01-01

To investigate the characteristics of expression and distribution of 5 subtypes of somatostatin receptors (SSTR1-5) in lung cancer, in situ hybridization was used to examine the expression patterns of SSTR mRNAs in 21 cases of different pathologic types of lung cancer tissues with [α- 35 S]dATP labelled oligonucleotides of the 5 SSTR subtypes as probes. Additionally, Leica Q-500 image analysis processing system was employed for the semi-quantitatively analysis of the hybridization signals. Patterns of SSTR1-5 expression in lung cancer tissues were found as follows. SSTR2 was prominent in small cell lung cancer (SCLC), whereas in non-small cell lung cancer (NSCLC) including the adenous cancer (Ad) and the squamous cancer (Sq), the expression of SSTR1 mRNA was stronger than that of the other 4 types. the expression density of SSTR1-5 in the NSCLC was higher that the SCLC (p < 0.01). The expression patterns and densities of the SSTR subtypes showed heterogeneity in different pathologic types of lung cancer. The expressions of the SSTR mRNAs in both SCLC and NSCLC indicated the positive prospects for somatostatin analog (SSA)-oriented agents in the treatment of both types of the lung cancer

Plaque assay for human coronavirus NL63 using human colon carcinoma cells

Directory of Open Access Journals (Sweden)

Drosten Christian

2008-11-01

Full Text Available Abstract Background Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen. Results 12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2 replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2. CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4th day of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL. Conclusion CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.

Potato spindle tuber viroid infection triggers degradation of chloride channel protein CLC-b-like and Ribosomal protein S3a-like mRNAs in tomato plants.

Science.gov (United States)

Adkar-Purushothama, Charith Raj; Iyer, Pavithran Sridharan; Perreault, Jean-Pierre

2017-08-21

It is well established that viroid derived small RNA (vd-sRNA) induces RNA silencing of endogenous mRNA. However, it remains not clear how exactly viroid infections can lead to severe symptom induction given the fact that fewer vd-sRNAs binding the specific target mRNAs were recovered from the infected plants. To answer this question, the two least expressed (+) and (-) strand vd-sRNAs of potato spindle tuber viroid (PSTVd) binding to both the 3' UTR and the coding region of tomato mRNAs were analyzed by infecting tomato plants with two variants of PSTVd. As products of these putative target mRNAs are involved in plant phenotype, the effect of this viroid on these genes were analyzed by infecting tomato plants with two variants of PSTVd. The direct interaction between the vd-sRNAs and putative mRNAs was validated by artificial microRNA experiments in a transient expression system and by RNA ligase-mediated rapid amplification of cDNA ends. Parallel analysis of RNA ends of viroid infected plants revealed the widespread cleavage of the target mRNAs in locations other than the vd-sRNA binding site during the viroid infection implying the viroid-infection induced vd-sRNA independent degradation of endogenous mRNAs during viroid infection.

Phenol emulsion-enhanced DNA-driven subtractive cDNA cloning: isolation of low-abundance monkey cortex-specific mRNAs

International Nuclear Information System (INIS)

Travis, G.H.; Sutcliffe, J.G.

1988-01-01

To isolate cDNA clones of low-abundance mRNAs expressed in monkey cerebral cortex but absent from cerebellum, the authors developed an improved subtractive cDNA cloning procedure that requires only modest quantities of mRNA. Plasmid DNA from a monkey cerebellum cDNA library was hybridized in large excess to radiolabeled monkey cortex cDNA in a phenol emulsion-enhanced reaction. The unhybridized cortex cDNA was isolated by chromatography on hydroxyapatite and used to probe colonies from a monkey cortex cDNA library. Of 60,000 colonies screened, 163 clones were isolated and confirmed by colony hybridization or RNA blotting to represent mRNAs, ranging from 0.001% to 0.1% abundance, specific to or highly enriched in cerebral cortex relative to cerebellum. Clones of one medium-abundance mRNA were recovered almost quantitatively. Two of the lower-abundance mRNAs were expressed at levels reduced by a factor of 10 in Alzheimer disease relative to normal human cortex. One of these was identified as the monkey preprosomatostatin I mRNA

Competitive fitness in coronaviruses is not correlated with size or number of double-membrane vesicles under reduced-temperature growth conditions.

Science.gov (United States)

Al-Mulla, Hawaa M N; Turrell, Lauren; Smith, Nicola M; Payne, Luke; Baliji, Surendranath; Züst, Roland; Thiel, Volker; Baker, Susan C; Siddell, Stuart G; Neuman, Benjamin W

2014-04-01

Positive-stranded viruses synthesize their RNA in membrane-bound organelles, but it is not clear how this benefits the virus or the host. For coronaviruses, these organelles take the form of double-membrane vesicles (DMVs) interconnected by a convoluted membrane network. We used electron microscopy to identify murine coronaviruses with mutations in nsp3 and nsp14 that replicated normally while producing only half the normal amount of DMVs under low-temperature growth conditions. Viruses with mutations in nsp5 and nsp16 produced small DMVs but also replicated normally. Quantitative reverse transcriptase PCR (RT-PCR) confirmed that the most strongly affected of these, the nsp3 mutant, produced more viral RNA than wild-type virus. Competitive growth assays were carried out in both continuous and primary cells to better understand the contribution of DMVs to viral fitness. Surprisingly, several viruses that produced fewer or smaller DMVs showed a higher fitness than wild-type virus at the reduced temperature, suggesting that larger and more numerous DMVs do not necessarily confer a competitive advantage in primary or continuous cell culture. For the first time, this directly demonstrates that replication and organelle formation may be, at least in part, studied separately during infection with positive-stranded RNA virus. IMPORTANCE The viruses that cause severe acute respiratory syndrome (SARS), poliomyelitis, and hepatitis C all replicate in double-membrane vesicles (DMVs). The big question about DMVs is why they exist in the first place. In this study, we looked at thousands of infected cells and identified two coronavirus mutants that made half as many organelles as normal and two others that made typical numbers but smaller organelles. Despite differences in DMV size and number, all four mutants replicated as efficiently as wild-type virus. To better understand the relative importance of replicative organelles, we carried out competitive fitness experiments. None

The 3'-terminal 55 nucleotides of bovine coronavirus defective interfering RNA harbor cis-acting elements required for both negative- and positive-strand RNA synthesis.

Directory of Open Access Journals (Sweden)

Wei-Yu Liao

Full Text Available The synthesis of the negative-strand [(--strand] complement of the ∼30 kilobase, positive-strand [(+-strand] coronaviral genome is a necessary early step for genome replication. The identification of cis-acting elements required for (--strand RNA synthesis in coronaviruses, however, has been hampered due to insufficiencies in the techniques used to detect the (--strand RNA species. Here, we employed a method of head-to-tail ligation and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR to detect and quantitate the synthesis of bovine coronavirus (BCoV defective interfering (DI RNA (- strands. Furthermore, using the aforementioned techniques along with Northern blot assay, we specifically defined the cis-acting RNA elements within the 3'-terminal 55 nucleotides (nts which function in the synthesis of (-- or (+-strand BCoV DI RNA. The major findings are as follows: (i nts from -5 to -39 within the 3'-terminal 55 nts are the cis-acting elements responsible for (--strand BCoV DI RNA synthesis, (ii nts from -3 to -34 within the 3'-terminal 55 nts are cis-acting elements required for (+-strand BCoV DI RNA synthesis, and (iii the nucleotide species at the 3'-most position (-1 is important, but not critical, for both (-- and (+-strand BCoV DI RNA synthesis. These results demonstrate that the 3'-terminal 55 nts in BCoV DI RNA harbor cis-acting RNA elements required for both (-- and (+-strand DI RNA synthesis and extend our knowledge on the mechanisms of coronavirus replication. The method of head-to-tail ligation and qRT-PCR employed in the study may also be applied to identify other cis-acting elements required for (--strand RNA synthesis in coronaviruses.

Pluripotent State Induction in Mouse Embryonic Fibroblast Using mRNAs of Reprogramming Factors

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Ahmed Kamel El-Sayed

2014-11-01

Full Text Available Reprogramming of somatic cells has great potential to provide therapeutic treatments for a number of diseases as well as provide insight into mechanisms underlying early embryonic development. Improvement of induced Pluripotent Stem Cells (iPSCs generation through mRNA-based methods is currently an area of intense research. This approach provides a number of advantages over previously used methods such as DNA integration and insertional mutagenesis. Using transfection of specifically synthesized mRNAs of various pluripotency factors, we generated iPSCs from mouse embryonic fibroblast (MEF cells. The genetic, epigenetic and functional properties of the iPSCs were evaluated at different times during the reprogramming process. We successfully introduced synthesized mRNAs, which localized correctly inside the cells and exhibited efficient and stable translation into proteins. Our work demonstrated a robust up-regulation and a gradual promoter de-methylation of the pluripotency markers, including non-transfected factors such as Nanog, SSEA-1 (stage-specific embryonic antigen 1 and Rex-1 (ZFP-42, zinc finger protein 42. Using embryonic stem cells (ESCs conditions to culture the iPS cells resulted in formation of ES-like colonies after approximately 12 days with only five daily repeated transfections. The colonies were positive for alkaline phosphatase and pluripotency-specific markers associated with ESCs. This study revealed the ability of pluripotency induction and generation of mouse mRNA induced pluripotent stem cells (mRNA iPSCs using transfection of specifically synthesized mRNAs of various pluripotency factors into mouse embryonic fibroblast (MEF cells. These generated iPSCs exhibited molecular and functional properties similar to ESCs, which indicate that this method is an efficient and viable alternative to ESCs and can be used for further biological, developmental and therapeutic investigations.

Kinetic models of gene expression including non-coding RNAs

Energy Technology Data Exchange (ETDEWEB)

Zhdanov, Vladimir P., E-mail: zhdanov@catalysis.r

2011-03-15

In cells, genes are transcribed into mRNAs, and the latter are translated into proteins. Due to the feedbacks between these processes, the kinetics of gene expression may be complex even in the simplest genetic networks. The corresponding models have already been reviewed in the literature. A new avenue in this field is related to the recognition that the conventional scenario of gene expression is fully applicable only to prokaryotes whose genomes consist of tightly packed protein-coding sequences. In eukaryotic cells, in contrast, such sequences are relatively rare, and the rest of the genome includes numerous transcript units representing non-coding RNAs (ncRNAs). During the past decade, it has become clear that such RNAs play a crucial role in gene expression and accordingly influence a multitude of cellular processes both in the normal state and during diseases. The numerous biological functions of ncRNAs are based primarily on their abilities to silence genes via pairing with a target mRNA and subsequently preventing its translation or facilitating degradation of the mRNA-ncRNA complex. Many other abilities of ncRNAs have been discovered as well. Our review is focused on the available kinetic models describing the mRNA, ncRNA and protein interplay. In particular, we systematically present the simplest models without kinetic feedbacks, models containing feedbacks and predicting bistability and oscillations in simple genetic networks, and models describing the effect of ncRNAs on complex genetic networks. Mathematically, the presentation is based primarily on temporal mean-field kinetic equations. The stochastic and spatio-temporal effects are also briefly discussed.

Transmissible gastroenteritis coronavirus genome packaging signal is located at the 5' end of the genome and promotes viral RNA incorporation into virions in a replication-independent process.

Science.gov (United States)

Morales, Lucia; Mateos-Gomez, Pedro A; Capiscol, Carmen; del Palacio, Lorena; Enjuanes, Luis; Sola, Isabel

2013-11-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5' end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3' end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies.

Genome-wide RIP-Chip analysis of translational repressor-bound mRNAs in the Plasmodium gametocyte

KAUST Repository

Guerreiro, Ana; Deligianni, Elena; Santos, Jorge M; Silva, Patricia AGC; Louis, Christos; Pain, Arnab; Janse, Chris J; Franke-Fayard, Blandine; Carret, Celine K; Siden-Kiamos, Inga; Mair, Gunnar R

2014-01-01

of RNA polymerase II transcription. Using GFP-tagging, we validate the repression phenotype of selected genes and identify mRNAs relying on the 5′ untranslated region for translational control. Gene deletion reveals a novel protein located in the ookinete

5'-Terminal AUGs in Escherichia coli mRNAs with Shine-Dalgarno Sequences: Identification and Analysis of Their Roles in Non-Canonical Translation Initiation.

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Heather J Beck

Full Text Available Analysis of the Escherichia coli transcriptome identified a unique subset of messenger RNAs (mRNAs that contain a conventional untranslated leader and Shine-Dalgarno (SD sequence upstream of the gene's start codon while also containing an AUG triplet at the mRNA's 5'- terminus (5'-uAUG. Fusion of the coding sequence specified by the 5'-terminal putative AUG start codon to a lacZ reporter gene, as well as primer extension inhibition assays, reveal that the majority of the 5'-terminal upstream open reading frames (5'-uORFs tested support some level of lacZ translation, indicating that these mRNAs can function both as leaderless and canonical SD-leadered mRNAs. Although some of the uORFs were expressed at low levels, others were expressed at levels close to that of the respective downstream genes and as high as the naturally leaderless cI mRNA of bacteriophage λ. These 5'-terminal uORFs potentially encode peptides of varying lengths, but their functions, if any, are unknown. In an effort to determine whether expression from the 5'-terminal uORFs impact expression of the immediately downstream cistron, we examined expression from the downstream coding sequence after mutations were introduced that inhibit efficient 5'-uORF translation. These mutations were found to affect expression from the downstream cistrons to varying degrees, suggesting that some 5'-uORFs may play roles in downstream regulation. Since the 5'-uAUGs found on these conventionally leadered mRNAs can function to bind ribosomes and initiate translation, this indicates that canonical mRNAs containing 5'-uAUGs should be examined for their potential to function also as leaderless mRNAs.

Increases of heat shock proteins and their mRNAs at high hydrostatic pressure in a deep-sea piezophilic bacterium, Shewanella violacea.

Science.gov (United States)

Sato, Hiroshi; Nakasone, Kaoru; Yoshida, Takao; Kato, Chiaki; Maruyama, Tadashi

2015-07-01

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

The NS1 Protein from Influenza Virus Stimulates Translation Initiation by Enhancing Ribosome Recruitment to mRNAs.

Science.gov (United States)

Panthu, Baptiste; Terrier, Olivier; Carron, Coralie; Traversier, Aurélien; Corbin, Antoine; Balvay, Laurent; Lina, Bruno; Rosa-Calatrava, Manuel; Ohlmann, Théophile

2017-10-27

The non-structural protein NS1 of influenza A viruses exerts pleiotropic functions during infection. Among these functions, NS1 was shown to be involved in the control of both viral and cellular translation; however, the mechanism by which this occurs remains to be determined. Thus, we have revisited the role of NS1 in translation by using a combination of influenza infection, mRNA reporter transfection, and in vitro functional and biochemical assays. Our data show that the NS1 protein is able to enhance the translation of virtually all tested mRNAs with the exception of constructs bearing the Dicistroviruses Internal ribosome entry segment (IRESes) (DCV and CrPV), suggesting a role at the level of translation initiation. The domain of NS1 required for translation stimulation was mapped to the RNA binding amino-terminal motif of the protein with residues R38 and K41 being critical for activity. Although we show that NS1 can bind directly to mRNAs, it does not correlate with its ability to stimulate translation. This activity rather relies on the property of NS1 to associate with ribosomes and to recruit them to target mRNAs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Feline infectious peritonitis: insights into feline coronavirus pathobiogenesis and epidemiology based on genetic analysis of the viral 3c gene.

Science.gov (United States)

Chang, Hui-Wen; de Groot, Raoul J; Egberink, Herman F; Rottier, Peter J M

2010-02-01

Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV), a virulent mutant of apathogenic feline enteric coronavirus (FECV). We analysed the 3c gene--a proposed virulence marker--in 27 FECV- and 28 FIPV-infected cats. Our findings suggest that functional 3c protein expression is crucial for FECV replication in the gut, but dispensable for systemic FIPV replication. Whilst intact in all FECVs, the 3c gene was mutated in the majority (71.4 %) of FIPVs, but not in all, implying that mutation in 3c is not the (single) cause of FIP. Most cats with FIP had no detectable intestinal feline coronaviruses (FCoVs) and had seemingly cleared the primary FECV infection. In those with detectable intestinal FCoV, the virus always had an intact 3c and seemed to have been acquired by FECV superinfection. Apparently, 3c-inactivated viruses replicate not at all--or only poorly--in the gut, explaining the rare incidence of FIP outbreaks.

Sialic Acid Binding Properties of Soluble Coronavirus Spike (S1 Proteins: Differences between Infectious Bronchitis Virus and Transmissible Gastroenteritis Virus

Directory of Open Access Journals (Sweden)

Christine Winter

2013-07-01

Full Text Available The spike proteins of a number of coronaviruses are able to bind to sialic acids present on the cell surface. The importance of this sialic acid binding ability during infection is, however, quite different. We compared the spike protein of transmissible gastroenteritis virus (TGEV and the spike protein of infectious bronchitis virus (IBV. Whereas sialic acid is the only receptor determinant known so far for IBV, TGEV requires interaction with its receptor aminopeptidase N to initiate infection of cells. Binding tests with soluble spike proteins carrying an IgG Fc-tag revealed pronounced differences between these two viral proteins. Binding of the IBV spike protein to host cells was in all experiments sialic acid dependent, whereas the soluble TGEV spike showed binding to APN but had no detectable sialic acid binding activity. Our results underline the different ways in which binding to sialoglycoconjugates is mediated by coronavirus spike proteins.

Structures of Two Coronavirus Main Proteases: Implications for Substrate Binding and Antiviral Drug Design

Energy Technology Data Exchange (ETDEWEB)

Xue, Xiaoyu; Yu, Hongwei; Yang, Haitao; Xue, Fei; Wu, Zhixin; Shen, Wei; Li, Jun; Zhou, Zhe; Ding, Yi; Zhao, Qi; Zhang, Xuejun C.; Liao, Ming; Bartlam, Mark; Rao, Zihe (SCAU); (Tsinghua); (Chinese Aca. Sci.)

2008-07-21

Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) MP{sup pro} and a severe acute respiratory syndrome CoV (SARS-CoV) M{sup pro} mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of M{sup pro}. A monomeric form of IBV M{sup pro} was identified for the first time in CoV M{sup pro} structures. A comparison of these two structures to other available M{sup pro} structures provides new insights for the design of substrate-based inhibitors targeting CoV M{sup pro}s. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV M{sup pro} and was found to demonstrate in vitro inactivation of IBV M{sup pro} and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV M{sup pro}.

Chimeric Feline Coronaviruses That Encode Type II Spike Protein on Type I Genetic Background Display Accelerated Viral Growth and Altered Receptor Usage▿

Science.gov (United States)

Tekes, Gergely; Hofmann-Lehmann, Regina; Bank-Wolf, Barbara; Maier, Reinhard; Thiel, Heinz-Jürgen; Thiel, Volker

2010-01-01

Persistent infection of domestic cats with feline coronaviruses (FCoVs) can lead to a highly lethal, immunopathological disease termed feline infectious peritonitis (FIP). Interestingly, there are two serotypes, type I and type II FCoVs, that can cause both persistent infection and FIP, even though their main determinant of host cell tropism, the spike (S) protein, is of different phylogeny and displays limited sequence identity. In cell culture, however, there are apparent differences. Type II FCoVs can be propagated to high titers by employing feline aminopeptidase N (fAPN) as a cellular receptor, whereas the propagation of type I FCoVs is usually difficult, and the involvement of fAPN as a receptor is controversial. In this study we have analyzed the phenotypes of recombinant FCoVs that are based on the genetic background of type I FCoV strain Black but encode the type II FCoV strain 79-1146 S protein. Our data demonstrate that recombinant FCoVs expressing a type II FCoV S protein acquire the ability to efficiently use fAPN for host cell entry and corroborate the notion that type I FCoVs use another main host cell receptor. We also observed that recombinant FCoVs display a large-plaque phenotype and, unexpectedly, accelerated growth kinetics indistinguishable from that of type II FCoV strain 79-1146. Thus, the main phenotypic differences for type I and type II FCoVs in cell culture, namely, the growth kinetics and the efficient usage of fAPN as a cellular receptor, can be attributed solely to the FCoV S protein. PMID:19906918

The sample of choice for detecting Middle East respiratory syndrome coronavirus in asymptomatic dromedary camels using real-time reversetranscription polymerase chain reaction.

NARCIS (Netherlands)

Mohran, K A; Farag, E A B; Reusken, C B E; Raj, V S; Lamers, M M; Pas, S D; Voermans, J; Smits, S L; Alhajri, M M; Alhajri, F; Al-Romaihi, H E; Ghobashy, H; El-Maghraby, M M; Al Dhahiry, S H S; Al-Mawlawi, N; El-Sayed, A M; Al-Thani, M; Al-Marri, S A; Haagmans, B L; Koopmans, M P G

2016-01-01

The newly identified Middle East respiratory syndrome coronavirus (MERS-CoV), which causes severe respiratory disease, particularly in people with comorbidities, requires further investigation. Studies in Qatar and elsewhere have provided evidence that dromedary camels are a reservoir for the virus,

Swine enteric coronavirus disease: A review of 4 years with porcine epidemic diarrhoea virus and porcine deltacoronavirus in the United States and Canada.

Science.gov (United States)

Niederwerder, M C; Hesse, R A

2018-06-01

Swine enteric coronaviruses, including porcine epidemic diarrhoea virus (PEDV) and porcine deltacoronavirus (PDCoV), have emerged and spread throughout the North American swine industry over the last four years. These diseases cause significant losses within the pork industry and within the first year after PEDV introduction, approximately 10% of the US herd died due to the disease. Similar to other enteric coronaviruses, such as transmissible gastroenteritis virus (TGEV), these emerging swine enteric coronavirus diseases (SECD) are age-dependent, with high morbidity and mortality in neonatal pigs. Since the introduction of SECD, research has focused on investigating viral pathogenesis through experimental inoculation, increasing maternal antibody for neonatal protection, understanding transmission risks through feed and transportation, and outlining the importance of biosecurity in preventing SECD introduction and spread. A survey of swine professionals conducted for this review revealed that the majority of respondents (75%) believe SECD can be eradicated and that most herds have been successful at long-term elimination of SECD after exposure (80%). However, unique properties of SECD, such as ineffective immunity through parenteral vaccination and a low oral infectious dose, play a major role in management of SECD. This review serves to describe the current knowledge of SECD and the characteristics of these viruses which provide both opportunities and challenges for long-term disease control and potential eradication from the US swine population. © 2018 Blackwell Verlag GmbH.

Activity-dependent expression of ELAV/Hu RBPs and neuronal mRNAs in seizure and cocaine brain.

Science.gov (United States)

Tiruchinapalli, Dhanrajan M; Caron, Marc G; Keene, Jack D

2008-12-01

Growing evidence indicates that both seizure (glutamate) and cocaine (dopamine) treatment modulate synaptic plasticity within the mesolimbic region of the CNS. Activation of glutamatergic neurons depends on the localized translation of neuronal mRNA products involved in modulating synaptic plasticity. In this study, we demonstrate the dendritic localization of HuR and HuD RNA-binding proteins (RBPs) and their association with neuronal mRNAs following these two paradigms of seizure and cocaine treatment. Both the ubiquitously expressed HuR and neuronal HuD RBPs were detected in different regions as well as within dendrites of the brain and in dissociated neurons. Quantitative analysis revealed an increase in HuR, HuD and p-glycogen synthase kinase 3beta (GSK3beta) protein levels as well as neuronal mRNAs encoding Homer, CaMKIIalpha, vascular early response gene, GAP-43, neuritin, and neuroligin protein products following either seizure or cocaine treatment. Inhibition of the Akt/GSK3beta signaling pathway by acute or chronic LiCl treatment revealed changes in HuR, HuD, pGSK3beta, p-Akt, and beta-catenin protein levels. In addition, a genetically engineered hyperdopaminergic mouse model (dopamine transporter knockout) revealed decreased expression of HuR protein levels, but no significant change was observed in HuD or fragile-X mental retardation protein RBPs. Finally, our data suggest that HuR and HuD RBPs potentially interact directly with neuronal mRNAs important for differentiation and synaptic plasticity.

Intracellular distribution of histone mRNAs in human fibroblasts studied by in situ hybridization

International Nuclear Information System (INIS)

Lawrence, J.B.; Singer, R.H.; Villnave, C.A.; Stein, J.L.; Stein, G.S.

1988-01-01

We have used in situ hybridization to study the intracellular distribution of mRNAs for cell cycle-dependent core and H1 histone proteins in human WI-38 fibroblasts. Because histones are abundant nuclear proteins and histone mRNA expression is tightly coupled to DNA synthesis, it was of interest to determine whether histone mRNAs are localized near the nucleus. Cells were hybridized with tritiated DNA probes specific for either histone H1, histone H4, actin, or poly(A)+ mRNA and were processed for autoradiography. In exponentially growing cultures, the fraction of histone mRNA-positive cells correlated well with the fraction of cells in S phase and was eliminated by hydroxyurea inhibition of DNA synthesis. Within individual cells the label for histone mRNA was widely distributed throughout the cytoplasm and did not appear to be more heavily concentrated near the nucleus. However, histone mRNA appeared to exhibit patchy, nonhomogeneous localization, and a quantitative evaluation confirmed that grain distributions were not as uniform as they were after hybridizations to poly(A)+ mRNA. Actin mRNA in WI-38 cells was also widely distributed throughout the cytoplasm but differed from histone mRNA in that label for actin mRNA was frequently most dense at the outermost region of narrow cell extensions. The localization of actin mRNA was less pronounced but qualitatively very similar to that previously described for chicken embryonic myoblasts and fibroblasts. We conclude that localization of histones in WI-38 cells is not facilitated by localization of histone protein synthesis near the nucleus and that there are subtle but discrete and potentially functional differences in the distributions of histone, actin, and poly(A)+ mRNAs

Genome-Wide Maps of m6A circRNAs Identify Widespread and Cell-Type-Specific Methylation Patterns that Are Distinct from mRNAs

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Chan Zhou

2017-08-01

Full Text Available N6-methyladenosine (m6A is the most abundant internal modification of mRNAs and is implicated in all aspects of post-transcriptional RNA metabolism. However, little is known about m6A modifications to circular (circ RNAs. We developed a computational pipeline (AutoCirc that, together with depletion of ribosomal RNA and m6A immunoprecipitation, defined thousands of m6A circRNAs with cell-type-specific expression. The presence of m6A circRNAs is corroborated by interaction between circRNAs and YTHDF1/YTHDF2, proteins that read m6A sites in mRNAs, and by reduced m6A levels upon depletion of METTL3, the m6A writer. Despite sharing m6A readers and writers, m6A circRNAs are frequently derived from exons that are not methylated in mRNAs, whereas mRNAs that are methylated on the same exons that compose m6A circRNAs exhibit less stability in a process regulated by YTHDF2. These results expand our understanding of the breadth of m6A modifications and uncover regulation of circRNAs through m6A modification.

Construction of recombinant Newcastle disease virus expressing the S1 protein of Turkey enteric coronavirus for use as a bivalent vaccine

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Turkey enteric coronavirus (TCoV) causes a contagious form of enteritis in turkeys, generally recognized in the field by outward signs including diarrhea and decreased weight gain, resulting in severe economic losses for the poultry industry in the US. To date there is no commercial vaccine availab...

Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus.

Science.gov (United States)

Jung, Kwonil; Alekseev, Konstantin P; Zhang, Xinsheng; Cheon, Doo-Sung; Vlasova, Anastasia N; Saif, Linda J

2007-12-01

The pathogenesis and optimal treatments for severe acute respiratory syndrome (SARS) are unclear, although corticosteroids were used to reduce lung and systemic inflammation. Because the pulmonary pathology of porcine respiratory coronavirus (PRCV) in pigs resembles SARS, we used PRCV as a model to clarify the effects of the corticosteroid dexamethasone (DEX) on coronavirus (CoV)-induced pneumonia. Conventional weaned pigs (n = 130) in one of four groups (PRCV/phosphate-buffered saline [PBS] [n = 41], PRCV/DEX [n = 41], mock/PBS [n = 23], and mock/DEX [n = 25]) were inoculated intranasally and intratracheally with the ISU-1 strain of PRCV (1 x 10(7) PFU) or cell culture medium. DEX was administered (once daily, 2 mg/kg of body weight/day, intramuscularly) from postinoculation day (PID) 1 to 6. In PRCV/DEX pigs, significantly milder pneumonia, fewer PRCV-positive cells, and lower viral RNA titers were present in lungs early at PID 2; however, at PID 4, 10, and 21, severe bronchointerstitial pneumonia, significantly higher numbers of PRCV-positive cells, and higher viral RNA titers were observed compared to results for PRCV/PBS pigs. Significantly lower numbers of CD2(+), CD3(+), CD4(+), and CD8(+) T cells were also observed in lungs of PRCV/DEX pigs than in those of PRCV/PBS pigs at PID 8 and 10, coincident with fewer gamma interferon (IFN-gamma)-secreting cells in the tracheobronchial lymph nodes as determined by enzyme-linked immunospot assay. Our results confirm that DEX treatment alleviates PRCV pneumonia early (PID 2) in the infection but continued use through PID 6 exacerbates later stages of infection (PID 4, 10, and 21), possibly by decreasing cellular immune responses in the lungs (IFN-gamma-secreting T cells), thereby creating an environment for more-extensive viral replication. These data have potential implications for corticosteroid use with SARS-CoV patients and suggest a precaution against prolonged use based on their unproven efficacy in humans

Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in adolescent male rat substantia nigra

Directory of Open Access Journals (Sweden)

Purves-Tyson Tertia D

2012-08-01

Full Text Available Abstract Background Increased risk of schizophrenia in adolescent males indicates that a link between the development of dopamine-related psychopathology and testosterone-driven brain changes may exist. However, contradictions as to whether testosterone increases or decreases dopamine neurotransmission are found and most studies address this in adult animals. Testosterone-dependent actions in neurons are direct via activation of androgen receptors (AR or indirect by conversion to 17β-estradiol and activation of estrogen receptors (ER. How midbrain dopamine neurons respond to sex steroids depends on the presence of sex steroid receptor(s and the level of steroid conversion enzymes (aromatase and 5α-reductase. We investigated whether gonadectomy and sex steroid replacement could influence dopamine levels by changing tyrosine hydroxylase (TH protein and mRNA and/or dopamine breakdown enzyme mRNA levels [catechol-O-methyl transferase (COMT and monoamine oxygenase (MAO A and B] in the adolescent male rat substantia nigra. We hypothesized that adolescent testosterone would regulate sex steroid signaling through regulation of ER and AR mRNAs and through modulation of aromatase and 5α-reductase mRNA levels. Results We find ERα and AR in midbrain dopamine neurons in adolescent male rats, indicating that dopamine neurons are poised to respond to circulating sex steroids. We report that androgens (T and DHT increase TH protein and increase COMT, MAOA and MAOB mRNAs in the adolescent male rat substantia nigra. We report that all three sex steroids increase AR mRNA. Differential action on ER pathways, with ERα mRNA down-regulation and ERβ mRNA up-regulation by testosterone was found. 5α reductase-1 mRNA was increased by AR activation, and aromatase mRNA was decreased by gonadectomy. Conclusions We conclude that increased testosterone at adolescence can shift the balance of sex steroid signaling to favor androgenic responses through promoting

Transmissible Gastroenteritis Coronavirus Genome Packaging Signal Is Located at the 5′ End of the Genome and Promotes Viral RNA Incorporation into Virions in a Replication-Independent Process

Science.gov (United States)

Morales, Lucia; Mateos-Gomez, Pedro A.; Capiscol, Carmen; del Palacio, Lorena; Sola, Isabel

2013-01-01

Preferential RNA packaging in coronaviruses involves the recognition of viral genomic RNA, a crucial process for viral particle morphogenesis mediated by RNA-specific sequences, known as packaging signals. An essential packaging signal component of transmissible gastroenteritis coronavirus (TGEV) has been further delimited to the first 598 nucleotides (nt) from the 5′ end of its RNA genome, by using recombinant viruses transcribing subgenomic mRNA that included potential packaging signals. The integrity of the entire sequence domain was necessary because deletion of any of the five structural motifs defined within this region abrogated specific packaging of this viral RNA. One of these RNA motifs was the stem-loop SL5, a highly conserved motif in coronaviruses located at nucleotide positions 106 to 136. Partial deletion or point mutations within this motif also abrogated packaging. Using TGEV-derived defective minigenomes replicated in trans by a helper virus, we have shown that TGEV RNA packaging is a replication-independent process. Furthermore, the last 494 nt of the genomic 3′ end were not essential for packaging, although this region increased packaging efficiency. TGEV RNA sequences identified as necessary for viral genome packaging were not sufficient to direct packaging of a heterologous sequence derived from the green fluorescent protein gene. These results indicated that TGEV genome packaging is a complex process involving many factors in addition to the identified RNA packaging signal. The identification of well-defined RNA motifs within the TGEV RNA genome that are essential for packaging will be useful for designing packaging-deficient biosafe coronavirus-derived vectors and providing new targets for antiviral therapies. PMID:23966403

Structure of a SARS coronavirus-derived peptide bound to the human major histocompatibility complex class I molecule HLA-B*1501

DEFF Research Database (Denmark)

Røder, Gustav; Kristensen, Ole; Kastrup, Jette S

2008-01-01

, the crystal structure of HLA-B*1501 in complex with a SARS coronavirus-derived nonapeptide (VQQESSFVM) has been determined at high resolution (1.87 A). The peptide is deeply anchored in the B and F pockets, but with the Glu4 residue pointing away from the floor in the peptide-binding groove, making...

Factors Influencing Emergency Nurses' Burnout During an Outbreak of Middle East Respiratory Syndrome Coronavirus in Korea

OpenAIRE

Kim, Ji Soo; Choi, Jeong Sil

2016-01-01

Purpose: Emergency department (ED) nurses suffer from persistent stress after experiencing the traumatic event of exposure to Middle East respiratory syndrome coronavirus (MERS-CoV), which can subsequently lead to burnout. This study aimed to assess ED nurses' burnout level during an outbreak of MERS-CoV and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. Methods: Study participants were ED nurses working in eight hosp...

Vaccines for emerging infectious diseases: Lessons from MERS coronavirus and Zika virus.

Science.gov (United States)

Maslow, Joel N

2017-12-02

The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed.

Evaluation of serologic and antigenic relationships between middle eastern respiratory syndrome coronavirus and other coronaviruses to develop vaccine platforms for the rapid response to emerging coronaviruses.

Science.gov (United States)

Agnihothram, Sudhakar; Gopal, Robin; Yount, Boyd L; Donaldson, Eric F; Menachery, Vineet D; Graham, Rachel L; Scobey, Trevor D; Gralinski, Lisa E; Denison, Mark R; Zambon, Maria; Baric, Ralph S

2014-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012, causing severe acute respiratory disease and pneumonia, with 44% mortality among 136 cases to date. Design of vaccines to limit the virus spread or diagnostic tests to track newly emerging strains requires knowledge of antigenic and serologic relationships between MERS-CoV and other CoVs.  Using synthetic genomics and Venezuelan equine encephalitis virus replicons (VRPs) expressing spike and nucleocapsid proteins from MERS-CoV and other human and bat CoVs, we characterize the antigenic responses (using Western blot and enzyme-linked immunosorbent assay) and serologic responses (using neutralization assays) against 2 MERS-CoV isolates in comparison with those of other human and bat CoVs.  Serologic and neutralization responses against the spike glycoprotein were primarily strain specific, with a very low level of cross-reactivity within or across subgroups. CoV N proteins within but not across subgroups share cross-reactive epitopes with MERS-CoV isolates. Our findings were validated using a convalescent-phase serum specimen from a patient infected with MERS-CoV (NA 01) and human antiserum against SARS-CoV, human CoV NL63, and human CoV OC43.  Vaccine design for emerging CoVs should involve chimeric spike protein containing neutralizing epitopes from multiple virus strains across subgroups to reduce immune pathology, and a diagnostic platform should include a panel of nucleocapsid and spike proteins from phylogenetically distinct CoVs.

Knowledge, Attitudes and Behaviours of Healthcare Workers in the Kingdom of Saudi Arabia to MERS Coronavirus and Other Emerging Infectious Diseases

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Abdullah J. Alsahafi

2016-12-01

Full Text Available Background: The Kingdom of Saudi Arabia has experienced a prolonged outbreak of Middle East Respiratory Syndrome (MERS coronavirus since 2012. Healthcare workers (HCWs form a significant risk group for infection. Objectives: The aim of this survey was to assess the knowledge, attitudes, infection control practices and educational needs of HCWs in the Kingdom of Saudi Arabia to MERS coronavirus and other emerging infectious diseases. Methods: 1500 of HCWs from Saudi Ministry of Health were invited to fill a questionnaire developed to cover the survey objectives from 9 September 2015 to 8 November 2015. The response rate was about 81%. Descriptive statistics was used to summarise the responses. Results: 1216 HCWs were included in this survey. A total of 56.5% were nurses and 22% were physicians. The most common sources of MERS-coronavirus (MERS-CoV information were the Ministry of Health (MOH memo (74.3%. Only (47.6% of the physicians, (30.4% of the nurses and (29.9% of the other HCWs were aware that asymptomatic MERS-CoV was described. Around half of respondents who having been investigated for MERS-CoV reported that their work performance decreased while they have suspicion of having MERS-CoV and almost two thirds reported having psychological problems during this period. Almost two thirds of the HCWs (61.2% reported anxiety about contracting MERS-CoV from patients. Conclusions: The knowledge about emerging infectious diseases was poor and there is need for further education and training programs particularly in the use of personal protective equipment, isolation and infection control measures. The self-reported infection control practices were sub-optimal and seem to be overestimated.

Isolation and characterization of current human coronavirus strains in primary human epithelial cell cultures reveal differences in target cell tropism

NARCIS (Netherlands)

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker; van der Hoek, Lia

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in

The effect of inhibition of PP1 and TNFα signaling on pathogenesis of SARS coronavirus

Energy Technology Data Exchange (ETDEWEB)

McDermott, Jason E.; Mitchell, Hugh D.; Gralinski, Lisa E.; Eisfeld, Amie J.; Josset, Laurence; Bankhead, Armand; Neumann, Gabriele; Tilton, Susan C.; Schäfer, Alexandra; Li, Chengjun; Fan, Shufang; McWeeney, Shannon; Baric, Ralph S.; Katze, Michael G.; Waters, Katrina M.

2016-09-23

The complex interplay between viral replication and host immune response during infection remains poorly understood. While many viruses are known to employ antiimmune strategies to facilitate their replication, highly pathogenic virus infections can also cause an excessive immune response that exacerbates, rather than reduces pathogenicity. To investigate this dichotomy in severe acute respiratory syndrome coronavirus (SARS-CoV), we developed a transcriptional network model of SARS-CoV infection in mice and used the model to prioritize candidate regulatory targets for further investigation. We validated our predictions in 18 different knockout (KO) mouse strains, showing that network topology provides significant predictive power to identify genes that are important for viral infection. We identified a novel player in the immune response to virus infection, Kepi, an inhibitory subunit of the protein phosphatase 1 (PP1) complex, which protects against SARS-CoV pathogenesis. We also found that receptors for the proinflammatory cytokine, tumor necrosis factor alpha (TNFα), promote pathogenesis through a parallel feed-forward circuit that promotes inflammation. These results are consistent with previous studies showing the role of over-stimulation of the inflammatory response to SARS-CoV in pathogenesis. We conclude that the critical balance between immune response and inflammation can be manipulated to improve the outcome of the infection. Further, our study provides two potential therapeutic strategies for mitigating the effects of SARS-CoV infection, and may provide insight into treatment strategies for Middle East Respiratory Syndrome Coronavirus (MERS-CoV).

Analyzing the interactions of mRNAs, miRNAs, lncRNAs and circRNAs to predict competing endogenous RNA networks in glioblastoma.

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Yuan, Yang; Jiaoming, Li; Xiang, Wang; Yanhui, Liu; Shu, Jiang; Maling, Gou; Qing, Mao

2018-05-01

Cross-talk between competitive endogenous RNAs (ceRNAs) may play a critical role in revealing potential mechanisms of tumor development and physiology. Glioblastoma is the most common type of malignant primary brain tumor, and the mechanisms of tumor genesis and development in glioblastoma are unclear. Here, to investigate the role of non-coding RNAs and the ceRNA network in glioblastoma, we performed paired-end RNA sequencing and microarray analyses to obtain the expression profiles of mRNAs, lncRNAs, circRNAs and miRNAs. We identified that the expression of 501 lncRNAs, 1999 mRNAs, 2038 circRNAs and 143 miRNAs were often altered between glioblastoma and matched normal brain tissue. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed on these differentially expressed mRNAs and miRNA-mediated target genes of lncRNAs and circRNAs. Furthermore, we used a multi-step computational framework and several bioinformatics methods to construct a ceRNA network combining mRNAs, miRNAs, lncRNAs and circRNA, based on co-expression analysis between the differentially expressed RNAs. We identified that plenty of lncRNAs, CircRNAs and their downstream target genes in the ceRNA network are related to glutamatergic synapse, suggesting that glutamate metabolism is involved in glioma biological functions. Our results will accelerate the understanding of tumorigenesis, cancer progression and even therapeutic targeting in glioblastoma.

Shine-dalgarno sequences play an essential role in the translation of plastid mRNAs in tobacco

DEFF Research Database (Denmark)

Scharff, Lars; Ehrnthaler, Miriam; Janowski, Marcin

2017-01-01

SD]). Although many chloroplast mRNAs harbor putative SDs in their 5' untranslated regions and the aSD displays strong conservation, the functional relevance of SD-aSD interactions in plastid translation is unclear. Here, by generating transplastomic tobacco (Nicotiana tabacum) mutants with point mutations...

Genotyping coronaviruses associated with feline infectious peritonitis.

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Lewis, Catherine S; Porter, Emily; Matthews, David; Kipar, Anja; Tasker, Séverine; Helps, Christopher R; Siddell, Stuart G

2015-06-01

Feline coronavirus (FCoV) infections are endemic among cats worldwide. The majority of infections are asymptomatic or result in only mild enteric disease. However, approximately 5 % of cases develop feline infectious peritonitis (FIP), a systemic disease that is a frequent cause of death in young cats. In this study, we report the complete coding genome sequences of six FCoVs: three from faecal samples from healthy cats and three from tissue lesion samples from cats with confirmed FIP. The six samples were obtained over a period of 8 weeks at a single-site cat rescue and rehoming centre in the UK. We found amino acid differences located at 44 positions across an alignment of the six virus translatomes and, at 21 of these positions, the differences fully or partially discriminated between the genomes derived from the faecal samples and the genomes derived from the tissue lesion samples. In this study, two amino acid differences fully discriminated the two classes of genomes: these were both located in the S2 domain of the virus surface glycoprotein gene. We also identified deletions in the 3c protein ORF of genomes from two of the FIP samples. Our results support previous studies that implicate S protein mutations in the pathogenesis of FIP. © 2015 The Authors.

Evidence for an Ancestral Association of Human Coronavirus 229E with Bats.

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Corman, Victor Max; Baldwin, Heather J; Tateno, Adriana Fumie; Zerbinati, Rodrigo Melim; Annan, Augustina; Owusu, Michael; Nkrumah, Evans Ewald; Maganga, Gael Darren; Oppong, Samuel; Adu-Sarkodie, Yaw; Vallo, Peter; da Silva Filho, Luiz Vicente Ribeiro Ferreira; Leroy, Eric M; Thiel, Volker; van der Hoek, Lia; Poon, Leo L M; Tschapka, Marco; Drosten, Christian; Drexler, Jan Felix

2015-12-01

We previously showed that close relatives of human coronavirus 229E (HCoV-229E) exist in African bats. The small sample and limited genomic characterizations have prevented further analyses so far. Here, we tested 2,087 fecal specimens from 11 bat species sampled in Ghana for HCoV-229E-related viruses by reverse transcription-PCR (RT-PCR). Only hipposiderid bats tested positive. To compare the genetic diversity of bat viruses and HCoV-229E, we tested historical isolates and diagnostic specimens sampled globally over 10 years. Bat viruses were 5- and 6-fold more diversified than HCoV-229E in the RNA-dependent RNA polymerase (RdRp) and spike genes. In phylogenetic analyses, HCoV-229E strains were monophyletic and not intermixed with animal viruses. Bat viruses formed three large clades in close and more distant sister relationships. A recently described 229E-related alpaca virus occupied an intermediate phylogenetic position between bat and human viruses. According to taxonomic criteria, human, alpaca, and bat viruses form a single CoV species showing evidence for multiple recombination events. HCoV-229E and the alpaca virus showed a major deletion in the spike S1 region compared to all bat viruses. Analyses of four full genomes from 229E-related bat CoVs revealed an eighth open reading frame (ORF8) located at the genomic 3' end. ORF8 also existed in the 229E-related alpaca virus. Reanalysis of HCoV-229E sequences showed a conserved transcription regulatory sequence preceding remnants of this ORF, suggesting its loss after acquisition of a 229E-related CoV by humans. These data suggested an evolutionary origin of 229E-related CoVs in hipposiderid bats, hypothetically with camelids as intermediate hosts preceding the establishment of HCoV-229E. The ancestral origins of major human coronaviruses (HCoVs) likely involve bat hosts. Here, we provide conclusive genetic evidence for an evolutionary origin of the common cold virus HCoV-229E in hipposiderid bats by analyzing a

Aberrant Expression of miRNA and mRNAs in Lesioned Tissues of Graves' Disease

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Qiu Qin

2015-03-01

Full Text Available Background and Aims: Abnormal microRNA (miRNA expression is found in many diseases including autoimmune diseases. However, little is known about the role of miRNA regulation in Graves' disease (GD. Here, we simultaneously detected different expressions of miRNA and mRNAs in thyroid tissues via a high-throughput transcriptomics approach, known as microarray, in order to reveal the relationship between aberrant expression of miRNAs and mRNAs spectrum and GD. Methods: Totally 7 specimens of thyroid tissue from 4 GD patients and 3 controls were obtained by surgery for microarray analysis. Then, 30 thyroid specimens (18 GD and 12 controls were also collected for further validation by quantitative real-time PCR ( qRT-PCR . Results: Statistical analysis showed that the expressions of 5 specific miRNA were increased significantly while those of other 18 miRNA were decreased in thyroid tissue of GD patients (FC≥1.3 or≤0.77 and pConclusion: Our study highlights the possibility that miRNA-target gene network may be involved in the pathogenesis of GD and could provide new insights into understanding the pathophysiological mechanisms of GD.

Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

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Picard Flora

2012-10-01

Full Text Available Abstract Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein

Vaccines for emerging infectious diseases: Lessons from MERS coronavirus and Zika virus

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Maslow, Joel N.

2017-01-01

ABSTRACT The past decade and a half has been characterized by numerous emerging infectious diseases. With each new threat, there has been a call for rapid vaccine development. Pathogens such as the Middle East Respiratory Syndrome coronavirus (MERS-CoV) and the Zika virus represent either new viral entities or viruses emergent in new geographic locales and characterized by novel complications. Both serve as paradigms for the global spread that can accompany new pathogens. In this paper, we review the epidemiology and pathogenesis of MERS-CoV and Zika virus with respect to vaccine development. The challenges in vaccine development and the approach to clinical trial design to test vaccine candidates for disease entities with a changing epidemiology are discussed. PMID:28846484

Development of an in situ hybridization assay for the detection of ostreid herpesvirus type 1 mRNAs in the Pacific oyster, Crassostrea gigas.

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Corbeil, Serge; Faury, Nicole; Segarra, Amélie; Renault, Tristan

2015-01-01

An in situ hybridization protocol for detecting mRNAs of ostreid herpesvirus type 1 (OsHV-1) which infects Pacific oysters, Crassostrea gigas, was developed. Three RNA probes were synthesized by cloning three partial OsHV-1 genes into plasmids using three specific primer pairs, and performing a transcription in the presence of digoxigenin dUTP. The RNA probes were able to detect the virus mRNAs in paraffin sections of experimentally infected oysters 26 h post-injection. The in situ hybridization showed that the OsHV-1 mRNAs were mainly present in connective tissues in gills, mantle, adductor muscle, digestive gland and gonads. DNA detection by in situ hybridization using a DNA probe and viral DNA quantitation by real-time PCR were also performed and results were compared with those obtained using RNA probes. Copyright © 2014 Elsevier B.V. All rights reserved.

Microarray-based identification and RT-PCR test screening for epithelial-specific mRNAs in peripheral blood of patients with colon cancer

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Coppola Domenico

2006-10-01

Full Text Available Abstract Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22 that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR. Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.

Molecular characterization of human coronaviruses and their circulation dynamics in Kenya, 2009-2012.

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Sipulwa, Lenata A; Ongus, Juliette R; Coldren, Rodney L; Bulimo, Wallace D

2016-02-01

Human Coronaviruses (HCoV) are a common cause of respiratory illnesses and are responsible for considerable morbidity and hospitalization across all age groups especially in individuals with compromised immunity. There are six known species of HCoV: HCoV-229E, HCoV-NL63, HCoV-HKU1, HCoV-OC43, MERS-CoV and SARS-HCoV. Although studies have shown evidence of global distribution of HCoVs, there is limited information on their presence and distribution in Kenya. HCoV strains that circulated in Kenya were retrospectively diagnosed and molecularly characterized. A total of 417 nasopharyngeal specimens obtained between January 2009 and December 2012 from around Kenya were analyzed by a real time RT-PCR using HCoV-specific primers. HCoV-positive specimens were subsequently inoculated onto monolayers of LL-CMK2 cells. The isolated viruses were characterized by RT-PCR amplification and sequencing of the partial polymerase (pol) gene. The prevalence of HCoV infection was as follows: out of the 417 specimens, 35 (8.4 %) were positive for HCoV, comprising 10 (2.4 %) HCoV-NL63, 12 (2.9 %) HCoV-OC43, 9 (2.1 %) HCoV-HKU1, and 4 (1 %) HCoV-229E. The Kenyan HCoV strains displayed high sequence homology to the prototypes and contemporaneous strains. Evolution analysis showed that the Kenyan HCoV-OC43 and HCoV-NL63 isolates were under purifying selection. Phylogenetic evolutionary analyses confirmed the identities of three HCoV-HKU1, five HCoV-NL63, eight HCoV-OC43 and three HCoV-229E. There were yearly variations in the prevalence and circulation patterns of individual HCoVs in Kenya. This paper reports on the first molecular characterization of human Coronaviruses in Kenya, which play an important role in causing acute respiratory infections among children.

Peptides corresponding to the predicted heptad repeat 2 domain of the feline coronavirus spike protein are potent inhibitors of viral infection.

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I-Jung Liu

Full Text Available BACKGROUND: Feline infectious peritonitis (FIP is a lethal immune-mediated disease caused by feline coronavirus (FCoV. Currently, no therapy with proven efficacy is available. In searching for agents that may prove clinically effective against FCoV infection, five analogous overlapping peptides were designed and synthesized based on the putative heptad repeat 2 (HR2 sequence of the spike protein of FCoV, and the antiviral efficacy was evaluated. METHODS: Plaque reduction assay and MTT (3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide cytotoxicity assay were performed in this study. Peptides were selected using a plaque reduction assay to inhibit Feline coronavirus infection. RESULTS: The results demonstrated that peptide (FP5 at concentrations below 20 μM inhibited viral replication by up to 97%. The peptide (FP5 exhibiting the most effective antiviral effect was further combined with a known anti-viral agent, human interferon-α (IFN-α, and a significant synergistic antiviral effect was observed. CONCLUSION: Our data suggest that the synthetic peptide FP5 could serve as a valuable addition to the current FIP prevention methods.

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

OpenAIRE

Gadir, Noga; Haim-Vilmovsky, Liora; Kraut-Cohen, Judith; Gerst, Jeffrey E.

2011-01-01

Targeted mRNA localization is a likely determinant of localized protein synthesis. To investigate whether mRNAs encoding mitochondrial proteins (mMPs) localize to mitochondria and, thus, might confer localized protein synthesis and import, we visualized endogenously expressed mMPs in vivo for the first time. We determined the localization of 24 yeast mMPs encoding proteins of the mitochondrial matrix, outer and inner membrane, and intermembrane space and found that many mMPs colocalize with m...

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia.

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Amer, Alazawy; Siti Suri, Arshad; Abdul Rahman, Omar; Mohd, Hair Bejo; Faruku, Bande; Saeed, Sharif; Tengku Azmi, Tengku Ibrahim

2012-11-21

Feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV) are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Of the total number of cats sampled, 95% (40/42) were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK), and Feline catus whole fetus-4 cells (Fcwf-4), show cytopathic effect (CPE) characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

Comparative in vivo analysis of recombinant type II feline coronaviruses with truncated and completed ORF3 region.

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Ádám Bálint

Full Text Available Our previous in vitro comparative study on a feline coronavirus (FCoV pair, differing only in the intactness of their ORF3abc regions, showed that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II feline infectious peritonitis virus (FIPV. In the present study, we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses. While parent virus FIPV DF-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus PBFIPV-DF-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original FIPV DF-2. PBFIPV-DF-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. The recombinant PBFIPV-DF-2-R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus (FECV and FIPV biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo.

Comparative In Vivo Analysis of Recombinant Type II Feline Coronaviruses with Truncated and Completed ORF3 Region

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Bálint, Ádám; Farsang, Attila; Zádori, Zoltán; Belák, Sándor

2014-01-01

Our previous in vitro comparative study on a feline coronavirus (FCoV) pair, differing only in the intactness of their ORF3abc regions, showed that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II feline infectious peritonitis virus (FIPV). In the present study, we describe a challenge experiment with the same recombinant FCoVs in order to gain data on the in vivo characteristics on these viruses. While parent virus FIPV DF-2 developed feline infectious peritonitis in all the infected cats, its recombinant virus PBFIPV-DF-2, differing only in seven nucleotides, proved to be surprisingly low virulent, although caused an acute febrile episode similarly to the original FIPV DF-2. PBFIPV-DF-2 infection induced significantly lower virus neutralization titers than its parent virus, and lacked the second phase of viremia and development of fatal course of the disease. The recombinant PBFIPV-DF-2-R3i with completed ORF3abc gained biological properties that differentiate between the feline enteric coronavirus (FECV) and FIPV biotypes such as intensive replication in the gut, absence of viremia and weak or no serological response. Using reverse genetic approaches our study is the first experimental proof that ORF3abc is indeed responsible for the restriction of FECV replication to the intestine in vivo. PMID:24586385

A human coronavirus responsible for the common cold massively kills dendritic cells but not monocytes.

Science.gov (United States)

Mesel-Lemoine, Mariana; Millet, Jean; Vidalain, Pierre-Olivier; Law, Helen; Vabret, Astrid; Lorin, Valérie; Escriou, Nicolas; Albert, Matthew L; Nal, Béatrice; Tangy, Frédéric

2012-07-01

Human coronaviruses are associated with upper respiratory tract infections that occasionally spread to the lungs and other organs. Although airway epithelial cells represent an important target for infection, the respiratory epithelium is also composed of an elaborate network of dendritic cells (DCs) that are essential sentinels of the immune system, sensing pathogens and presenting foreign antigens to T lymphocytes. In this report, we show that in vitro infection by human coronavirus 229E (HCoV-229E) induces massive cytopathic effects in DCs, including the formation of large syncytia and cell death within only few hours. In contrast, monocytes are much more resistant to infection and cytopathic effects despite similar expression levels of CD13, the membrane receptor for HCoV-229E. While the differentiation of monocytes into DCs in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4 requires 5 days, only 24 h are sufficient for these cytokines to sensitize monocytes to cell death and cytopathic effects when infected by HCoV-229E. Cell death induced by HCoV-229E is independent of TRAIL, FasL, tumor necrosis factor alpha, and caspase activity, indicating that viral replication is directly responsible for the observed cytopathic effects. The consequence of DC death at the early stage of HCoV-229E infection may have an impact on the early control of viral dissemination and on the establishment of long-lasting immune memory, since people can be reinfected multiple times by HCoV-229E.

Suppression of feline coronavirus replication in vitro by cyclosporin A

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Tanaka Yoshikazu

2012-04-01

Full Text Available Abstract The feline infectious peritonitis virus (FIPV is a member of the feline coronavirus family that causes FIP, which is incurable and fatal in cats. Cyclosporin A (CsA, an immunosuppressive agent that targets the nuclear factor pathway of activated T-cells (NF-AT to bind cellular cyclophilins (CyP, dose-dependently inhibited FIPV replication in vitro. FK506 (an immunosuppressor of the pathway that binds cellular FK506-binding protein (FKBP but not CyP did not affect FIPV replication. Neither cell growth nor viability changed in the presence of either CsA or FK506, and these factors did not affect the NF-AT pathway in fcwf-4 cells. Therefore, CsA does not seem to exert inhibitory effects via the NF-AT pathway. In conclusion, CsA inhibited FIPV replication in vitro and further studies are needed to verify the practical value of CsA as an anti-FIPV treatment in vivo.

A case of imported Middle East Respiratory Syndrome coronavirus infection and public health response, Greece, April 2014.

Science.gov (United States)

Tsiodras, S; Baka, A; Mentis, A; Iliopoulos, D; Dedoukou, X; Papamavrou, G; Karadima, S; Emmanouil, M; Kossyvakis, A; Spanakis, N; Pavli, A; Maltezou, H; Karageorgou, A; Spala, G; Pitiriga, V; Kosmas, E; Tsiagklis, S; Gkatzias, S; Koulouris, Ng; Koutsoukou, A; Bakakos, P; Markozanhs, E; Dionellis, G; Pontikis, K; Rovina, N; Kyriakopoulou, M; Efstathiou, P; Papadimitriou, T; Kremastinou, J; Tsakris, A; Saroglou, G

2014-04-24

On 18 April 2014, a case of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infection was laboratory confirmed in Athens, Greece in a patient returning from Jeddah, Saudi Arabia. Main symptoms upon initial presentation were protracted fever and diarrhoea, during hospitalisation he developed bilateral pneumonia and his condition worsened. During 14 days prior to onset of illness, he had extensive contact with the healthcare environment in Jeddah. Contact tracing revealed 73 contacts, no secondary cases had occurred by 22 April.

Real-time sequence-validated loop-mediated isothermal amplification assays for detection of Middle East respiratory syndrome coronavirus (MERS-CoV.

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Sanchita Bhadra

Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV, an emerging human coronavirus, causes severe acute respiratory illness with a 35% mortality rate. In light of the recent surge in reported infections we have developed asymmetric five-primer reverse transcription loop-mediated isothermal amplification (RT-LAMP assays for detection of MERS-CoV. Isothermal amplification assays will facilitate the development of portable point-of-care diagnostics that are crucial for management of emerging infections. The RT-LAMP assays are designed to amplify MERS-CoV genomic loci located within the open reading frame (ORF1a and ORF1b genes and upstream of the E gene. Additionally we applied one-step strand displacement probes (OSD for real-time sequence-specific verification of LAMP amplicons. Asymmetric amplification effected by incorporating a single loop primer in each assay accelerated the time-to-result of the OSD-RT-LAMP assays. The resulting assays could detect 0.02 to 0.2 plaque forming units (PFU (5 to 50 PFU/ml of MERS-CoV in infected cell culture supernatants within 30 to 50 min and did not cross-react with common human respiratory pathogens.

Optimized Mitochondrial Targeting of Proteins Encoded by Modified mRNAs Rescues Cells Harboring Mutations in mtATP6

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Randall Marcelo Chin

2018-03-01

Full Text Available Summary: Mitochondrial disease may be caused by mutations in the protein-coding genes of the mitochondrial genome. A promising strategy for treating such diseases is allotopic expression—the translation of wild-type copies of these proteins in the cytosol, with subsequent translocation into the mitochondria, resulting in rescue of mitochondrial function. In this paper, we develop an automated, quantitative, and unbiased screening platform to evaluate protein localization and mitochondrial morphology. This platform was used to compare 31 mitochondrial targeting sequences and 15 3′ UTRs in their ability to localize up to 9 allotopically expressed proteins to the mitochondria and their subsequent impact on mitochondrial morphology. Taking these two factors together, we synthesized chemically modified mRNAs that encode for an optimized allotopic expression construct for mtATP6. These mRNAs were able to functionally rescue a cell line harboring the 8993T > G point mutation in the mtATP6 gene. : Allotopic expression of proteins normally encoded by mtDNA is a promising therapy for mitochondrial disease. Chin et al. use an unbiased and high-content imaging-based screening platform to optimize allotopic expression. Modified mRNAs encoding for the optimized allotopic expression constructs rescued the respiration and growth of mtATP6-deficient cells. Keywords: mitochondria, mitochondrial disease, mRNA, modified mRNA, ATP6, allotopic expression, rare disease, gene therapy, screening, high content imaging

The use of enzyme-linked immunosorbent assay systems for the serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

NARCIS (Netherlands)

G.F. Rimmelzwaan (Guus); J. Groen (Jan); H.F. Egberink (Herman); G.H.A. Borst (Gerrit); F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

1991-01-01

textabstractComplex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-,

The Fragile X Protein binds mRNAs involved in cancer progression and modulates metastasis formation

Science.gov (United States)

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; Fata, Giorgio La; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-01-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. PMID:24092663

Dynamics of SARS-coronavirus HR2 domain in the prefusion and transition states

Science.gov (United States)

McReynolds, Susanna; Jiang, Shaokai; Rong, Lijun; Caffrey, Michael

2009-12-01

The envelope glycoproteins S1 and S2 of severe acute respiratory syndrome coronavirus (SARS-CoV) mediate viral entry by conformational change from a prefusion state to a postfusion state that enables fusion of the viral and target membranes. In this work we present the characterization of the dynamic properties of the SARS-CoV S2-HR2 domain (residues 1141-1193 of S) in the prefusion and newly discovered transition states by NMR 15N relaxation studies. The dynamic properties of the different states, which are stabilized under different experimental conditions, extend the current model of viral membrane fusion and give insight into the design of structure-based antagonists of SARS-CoV in particular, as well as other enveloped viruses such as HIV.

In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

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Jeremiah Athmer

2017-01-01

Full Text Available Coronavirus (CoV replication and transcription are carried out in close proximity to restructured endoplasmic reticulum (ER membranes in replication/transcription complexes (RTC. Many of the CoV nonstructural proteins (nsps are required for RTC function; however, not all of their functions are known. nsp15 contains an endoribonuclease domain that is conserved in the CoV family. While the enzymatic activity and crystal structure of nsp15 are well defined, its role in replication remains elusive. nsp15 localizes to sites of RNA replication, but whether it acts independently or requires additional interactions for its function remains unknown. To begin to address these questions, we created an in situ tagged form of nsp15 using the prototypic CoV, mouse hepatitis virus (MHV. In MHV, nsp15 contains the genomic RNA packaging signal (P/S, a 95-bp RNA stem-loop structure that is not required for viral replication or nsp15 function. Utilizing this knowledge, we constructed an internal hemagglutinin (HA tag that replaced the P/S. We found that nsp15-HA was localized to discrete perinuclear puncta and strongly colocalized with nsp8 and nsp12, both well-defined members of the RTC, but not the membrane (M protein, involved in virus assembly. Finally, we found that nsp15 interacted with RTC-associated proteins nsp8 and nsp12 during infection, and this interaction was RNA independent. From this, we conclude that nsp15 localizes and interacts with CoV proteins in the RTC, suggesting it plays a direct or indirect role in virus replication. Furthermore, the use of in situ epitope tags could be used to determine novel nsp-nsp interactions in coronaviruses.

Isolation and molecular characterization of type I and type II feline coronavirus in Malaysia

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Amer Alazawy

2012-11-01

Full Text Available Abstract Background Feline infectious peritonitis virus (FIPV and feline enteric coronavirus (FECV are two important coronaviruses of domestic cat worldwide. Although FCoV is prevalent among cats; the fastidious nature of type I FCoV to grow on cell culture has limited further studies on tissue tropism and pathogenesis of FCoV. While several studies reported serological evidence for FCoV in Malaysia, neither the circulating FCoV isolated nor its biotypes determined. This study for the first time, describes the isolation and biotypes determination of type I and type II FCoV from naturally infected cats in Malaysia. Findings Of the total number of cats sampled, 95% (40/42 were RT-PCR positive for FCoV. Inoculation of clinical samples into Crandell feline kidney cells (CrFK, and Feline catus whole fetus-4 cells (Fcwf-4, show cytopathic effect (CPE characterized by syncytial cells formation and later cell detachment. Differentiation of FCoV biotypes using RT-PCR assay revealed that, 97.5% and 2.5% of local isolates were type I and type II FCoV, respectively. These isolates had high sequence homology and phylogenetic similarity with several FCoV isolates from Europe, South East Asia and USA. Conclusions This study reported the successful isolation of local type I and type II FCoV evident with formation of cytopathic effects in two types of cell cultures namely the CrFK and Fcwf-4 , where the later cells being more permissive. However, the RT-PCR assay is more sensitive in detecting the antigen in suspected samples as compared to virus isolation in cell culture. The present study indicated that type I FCoV is more prevalent among cats in Malaysia.

Middle East respiratory syndrome coronavirus disease is rare in children: An update from Saudi Arabia.

Science.gov (United States)

Al-Tawfiq, Jaffar A; Kattan, Rana F; Memish, Ziad A

2016-11-08

To summarize the reported Middle East respiratory syndrome-coronavirus (MERS-CoV) cases, the associated clinical presentations and the outcomes. We searched the Saudi Ministry of Health website, the World Health Organization website, and the Flutracker website. We also searched MEDLINE and PubMed for the keywords: Middle East respiratory syndrome-coronavirus, MERS-CoV in combination with pediatric, children, childhood, infancy and pregnancy from the initial discovery of the virus in 2012 to 2016. The retrieved articles were also read to further find other articles. Relevant data were placed into an excel sheet and analyzed accordingly. Descriptive analytic statistics were used in the final analysis as deemed necessary. From June 2012 to April 19, 2016, there were a total of 31 pediatric MERS-CoV cases. Of these cases 13 (42%) were asymptomatic and the male to female ratio was 1.7:1. The mean age of patients was 9.8 ± 5.4 years. Twenty-five (80.6%) of the cases were reported from the Kingdom of Saudi Arabia. The most common source of infection was household contact (10 of 15 with reported source) and 5 patients acquired infection within a health care facility. Using real time reverse transcriptase polymerase chain reaction of pediatric patients revealed that 9 out of 552 (1.6%) was positive in the Kingdom of Saudi Arabia. Utilizing serology for MERS-CoV infection in Jordan and Saudi Arabia did not reveal any positive patients. Thus, the number of the pediatric MERS-CoV is low; the exact reason for the low prevalence of the disease in children is not known.

Severe acute respiratory syndrome--a new coronavirus from the Chinese dragon's lair

DEFF Research Database (Denmark)

Knudsen, T B; Kledal, T N; Andersen, O

2003-01-01

Health Organization (WHO). As SARS has the potential of becoming the first pandemic of the new millennium, a global warning by the WHO was issued on 12 March 2003. The disease, which is believed to have its origin in the Chinese Guangdong province, spread from Hong Kong via international airports to its...... disease constitutes an unprecedented scientific achievement. The main scope of the article is to provide the clinician with an overview of the natural history, epidemiology and clinical characteristics of SARS. On the basis of the recently published viral genome and structural features common...... to the members of the coronavirus family, a model for host cell-virus interaction and possible targets for antiviral drugs are presented. The epidemiological consequences of introducing a novel pathogen in a previously unexposed population and the origin and evolution of a new and more pathogenic strain...

Candidates in Astroviruses, Seadornaviruses, Cytorhabdoviruses and Coronaviruses for +1 frame overlapping genes accessed by leaky scanning

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Atkins John F

2010-01-01

Full Text Available Abstract Background Overlapping genes are common in RNA viruses where they serve as a mechanism to optimize the coding potential of compact genomes. However, annotation of overlapping genes can be difficult using conventional gene-finding software. Recently we have been using a number of complementary approaches to systematically identify previously undetected overlapping genes in RNA virus genomes. In this article we gather together a number of promising candidate new overlapping genes that may be of interest to the community. Results Overlapping gene predictions are presented for the astroviruses, seadornaviruses, cytorhabdoviruses and coronaviruses (families Astroviridae, Reoviridae, Rhabdoviridae and Coronaviridae, respectively.

Effects of pathogen reduction systems on platelet microRNAs, mRNAs, activation, and function.

Science.gov (United States)

Osman, Abdimajid; Hitzler, Walter E; Meyer, Claudius U; Landry, Patricia; Corduan, Aurélie; Laffont, Benoit; Boilard, Eric; Hellstern, Peter; Vamvakas, Eleftherios C; Provost, Patrick

2015-01-01

Pathogen reduction (PR) systems for platelets, based on chemically induced cross-linking and inactivation of nucleic acids, potentially prevent transfusion transmission of infectious agents, but can increase clinically significant bleeding in some clinical studies. Here, we documented the effects of PR systems on microRNA and mRNA levels of platelets stored in the blood bank, and assessed their impact on platelet activation and function. Unlike platelets subjected to gamma irradiation or stored in additive solution, platelets treated with Intercept (amotosalen+ ultraviolet-A [UVA] light) exhibited significantly reduced levels of 6 of the 11 microRNAs, and 2 of the 3 anti-apoptotic mRNAs (Bcl-xl and Clusterin) that we monitored, compared with platelets stored in plasma. Mirasol (riboflavin+ UVB light) treatment of platelets did not produce these effects. PR neither affected platelet microRNA synthesis or function nor induced cross-linking of microRNA-sized endogenous platelet RNA species. However, the reduction in the platelet microRNA levels induced by Intercept correlated with the platelet activation (p < 0.05) and an impaired platelet aggregation response to ADP (p < 0.05). These results suggest that Intercept treatment may induce platelet activation, resulting in the release of microRNAs and mRNAs from platelets. The clinical implications of this reduction in platelet nucleic acids secondary to Intercept remain to be established.

Coronavirus and influenza virus proteolytic priming takes place in tetraspanin-enriched membrane microdomains.

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Earnest, James T; Hantak, Michael P; Park, Jung-Eun; Gallagher, Tom

2015-06-01

Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus

Interplay of noncoding RNAs, mRNAs, and proteins during the growth of eukaryotic cells

International Nuclear Information System (INIS)

Zhdanov, V. P.

2010-01-01

Numerous biological functions of noncoding RNAs (ncRNAs) in eukaryotic cells are based primarily on their ability to pair with target mRNAs and then either to prevent translation or to result in rapid degradation of the mRNA-ncRNA complex. Using a general model describing this scenario, we show that ncRNAs may help to maintain constant mRNA and protein concentrations during the growth of cells. The possibility of observation of this effect on the global scale is briefly discussed.

The fragile X protein binds mRNAs involved in cancer progression and modulates metastasis formation.

Science.gov (United States)

Lucá, Rossella; Averna, Michele; Zalfa, Francesca; Vecchi, Manuela; Bianchi, Fabrizio; La Fata, Giorgio; Del Nonno, Franca; Nardacci, Roberta; Bianchi, Marco; Nuciforo, Paolo; Munck, Sebastian; Parrella, Paola; Moura, Rute; Signori, Emanuela; Alston, Robert; Kuchnio, Anna; Farace, Maria Giulia; Fazio, Vito Michele; Piacentini, Mauro; De Strooper, Bart; Achsel, Tilmann; Neri, Giovanni; Neven, Patrick; Evans, D Gareth; Carmeliet, Peter; Mazzone, Massimiliano; Bagni, Claudia

2013-10-01

The role of the fragile X mental retardation protein (FMRP) is well established in brain, where its absence leads to the fragile X syndrome (FXS). FMRP is almost ubiquitously expressed, suggesting that, in addition to its effects in brain, it may have fundamental roles in other organs. There is evidence that FMRP expression can be linked to cancer. FMR1 mRNA, encoding FMRP, is overexpressed in hepatocellular carcinoma cells. A decreased risk of cancer has been reported in patients with FXS while a patient-case with FXS showed an unusual decrease of tumour brain invasiveness. However, a role for FMRP in regulating cancer biology, if any, remains unknown. We show here that FMRP and FMR1 mRNA levels correlate with prognostic indicators of aggressive breast cancer, lung metastases probability and triple negative breast cancer (TNBC). We establish that FMRP overexpression in murine breast primary tumours enhances lung metastasis while its reduction has the opposite effect regulating cell spreading and invasion. FMRP binds mRNAs involved in epithelial mesenchymal transition (EMT) and invasion including E-cadherin and Vimentin mRNAs, hallmarks of EMT and cancer progression. © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.

Middle East respiratory syndrome coronavirus (MERS-CoV) RNA and neutralising antibodies in milk collected according to local customs from dromedary camels, Qatar, April 2014

NARCIS (Netherlands)

Reusken, C B; Farag, E A; Jonges, M; Godeke, G J; El-Sayed, A M; Pas, S D; Raj, V S; Mohran, K A; Moussa, H A; Ghobashy, H; Alhajri, F; Ibrahim, A K; Bosch, B J; Pasha, S K; Al-Romaihi, H E; Al-Thani, M; Al-Marri, S A; AlHajri, M M; Haagmans, B L; Koopmans, M P

2014-01-01

Antibodies to Middle East respiratory syndrome coronavirus (MERS-CoV) were detected in serum and milk collected according to local customs from 33 camels in Qatar, April 2014. At one location, evidence for active virus shedding in nasal secretions and/or faeces was observed for 7/12 camels; viral

Feline infectious peritonitis: role of the feline coronavirus 3c gene in intestinal tropism and pathogenicity based upon isolates from resident and adopted shelter cats.

Science.gov (United States)

Pedersen, Niels C; Liu, Hongwei; Scarlett, Jennifer; Leutenegger, Christian M; Golovko, Lyudmila; Kennedy, Heather; Kamal, Farina Mustaffa

2012-04-01

Feline infectious peritonitis virus (FIPV) was presumed to arise from mutations in the 3c of a ubiquitous and largely nonpathogenic feline enteric coronavirus (FECV). However, a recent study found that one-third of FIPV isolates have an intact 3c and suggested that it is not solely involved in FIP but is essential for intestinal replication. In order to confirm these assumptions, 27 fecal and 32 FIP coronavirus isolates were obtained from resident or adopted cats from a large metropolitan shelter during 2008-2009 and their 3a-c, E, and M genes sequenced. Forty percent of coronavirus isolates from FIP tissues had an intact 3c gene, while 60% had mutations that truncated the gene product. The 3c genes of fecal isolates from healthy cats were always intact. Coronavirus from FIP diseased tissues consistently induced FIP when given either oronasally or intraperitoneally (i.p.), regardless of the functional status of their 3c genes, thus confirming them to be FIPVs. In contrast, fecal isolates from healthy cats were infectious following oronasal infection and shed at high levels in feces without causing disease, as expected for FECVs. Only one in three cats shed FECV in the feces following i.p. infection, indicating that FECVs can replicate systemically, but with difficulty. FIPVs having a mutated 3c were not shed in the feces following either oronasal or i.p. inoculation, while FIPVs with intact 3c genes were shed in the feces following oronasal but not i.p. inoculation. Therefore, an intact 3c appears to be essential for intestinal replication. Although FIPVs with an intact 3c were shed in the feces following oronasal inoculation, fecal virus from these cats was not infectious for other cats. Attempts to identify potential FIP mutations in the 3a, 3b, E, and M were negative. However, the 3c gene of FIPVs, even though appearing intact, contained many more non-synonymous amino acid changes in the 3' one-third of the 3c protein than FECVs. An attempt to trace FIPV

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

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Yin, Wanzhong; Wang, Ping; Wang, Xin [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China); Song, Wenzhi [Department of Stomatology, China-Japan Friendship Hospital, Jilin University, Changchun (China); Cui, Xiangyan; Yu, Hong; Zhu, Wei [Department of Otorhinolaryngology, Head and Neck Surgery, The First Clinical Hospital, Norman Bethune College of Medicine, Jilin University, Changchun (China)

2013-06-12

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.

Identification of microRNAs and mRNAs associated with multidrug resistance of human laryngeal cancer Hep-2 cells

International Nuclear Information System (INIS)

Yin, Wanzhong; Wang, Ping; Wang, Xin; Song, Wenzhi; Cui, Xiangyan; Yu, Hong; Zhu, Wei

2013-01-01

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer

False-Positive Results in a Recombinant Severe Acute Respiratory Syndrome-Associated Coronavirus (SARS-CoV) Nucleocapsid Enzyme-Linked Immunosorbent Assay Due to HCoV-OC43 and HCoV-229E Rectified by Western Blotting with Recombinant SARS-CoV Spike Polypeptide

OpenAIRE

Woo, Patrick C. Y.; Lau, Susanna K. P.; Wong, Beatrice H. L.; Chan, Kwok-Hung; Hui, Wai-Ting; Kwan, Grace S. W.; Peiris, J. S. Malik; Couch, Robert B.; Yuen, Kwok-Yung

2004-01-01

Using paired serum samples obtained from patients with illness associated with increases in anti-human coronavirus OC43 (HCoV-OC43) or anti-HCoV-229E antibodies, we examined the possibility of false-positive results detected in a recombinant severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) nucleocapsid protein immunoglobulin G enzyme-linked immunosorbent assay (ELISA). Three of the 21 and 1 of the 7 convalescent-phase serum samples from persons with increases in anti...

Identification of mRNAs that move over long distances using an RNA-Seq analysis of Arabidopsis/Nicotiana benthamiana heterografts.

Science.gov (United States)

Notaguchi, Michitaka; Higashiyama, Tetsuya; Suzuki, Takamasa

2015-02-01

Phloem is a conductive tissue that allocates nutrients from mature source leaves to sinks such as young developing tissues. Phloem also delivers proteins and RNA species, such as small RNAs and mRNAs. Intensive studies on plant systemic signaling revealed the essential roles of proteins and RNA species. However, many of their functions are still largely unknown, with the roles of transported mRNAs being particularly poorly understood. A major difficulty is the absence of an accurate and comprehensive list of mobile transcripts. In this study, we used a hetero-graft system with Nicotiana benthamiana as the recipient scion and Arabidopsis as the donor stock, to identify transcripts that moved long distances across the graft union. We identified 138 Arabidopsis transcripts as mobile mRNAs, which we collectively termed the mRNA mobilome. Reverse transcription-PCR, quantitative real-time PCR and droplet digital PCR analyses confirmed the mobility. The transcripts included potential signaling factors and, unexpectedly, more general factors. In our investigations, we found no preferred transcript length, no previously known sequence motifs in promoter or transcript sequences and no similarities between the level of the transcripts and that in the source leaves. Grafting experiments regarding the function of ERECTA, an identified transcript, showed that no function of the transcript mobilized. To our knowledge, this is the first report identifying transcripts that move over long distances using a hetero-graft system between different plant taxa. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

No evidence that mRNAs have lower folding free energies than random sequences with the same dinucleotide distribution

DEFF Research Database (Denmark)

Workman, Christopher; Krogh, Anders Stærmose

1999-01-01

This work investigates whether mRNA has a lower estimated folding free energy than random sequences. The free energy estimates are calculated by the mfold program for prediction of RNA secondary structures. For a set of 46 mRNAs it is shown that the predicted free energy is not significantly diff...

Stress and Withdrawal from Chronic Ethanol Induce Selective Changes in Neuroimmune mRNAs in Differing Brain Sites

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Darin J. Knapp

2016-07-01

Full Text Available Stress is a strong risk factor in alcoholic relapse and may exert effects that mimic aspects of chronic alcohol exposure on neurobiological systems. With the neuroimmune system becoming a prominent focus in the study of the neurobiological consequences of stress, as well as chronic alcohol exposure proving to be a valuable focus in this regard, the present study sought to compare the effects of stress and chronic ethanol exposure on induction of components of the neuroimmune system. Rats were exposed to either 1 h exposure to a mild stressor (restraint or exposure to withdrawal from 15 days of chronic alcohol exposure (i.e., withdrawal from chronic ethanol, WCE and assessed for neuroimmune mRNAs in brain. Restraint stress alone elevated chemokine (C–C motif ligand 2 (CCL2, interleukin-1-beta (IL-1β, tumor necrosis factor alpha (TNFα and toll-like receptor 4 (TLR4 mRNAs in the cerebral cortex within 4 h with a return to a control level by 24 h. These increases were not accompanied by an increase in corresponding proteins. Withdrawal from WCE also elevated cytokines, but did so to varying degrees across different cytokines and brain regions. In the cortex, stress and WCE induced CCL2, TNFα, IL-1β, and TLR4 mRNAs. In the hypothalamus, only WCE induced cytokines (CCL2 and IL-1β while in the hippocampus, WCE strongly induced CCL2 while stress and WCE induced IL-1β. In the amygdala, only WCE induced CCL2. Finally—based on the previously demonstrated role of corticotropin-releasing factor 1 (CRF1 receptor inhibition in blocking WCE-induced cytokine mRNAs—the CRF1 receptor antagonist CP154,526 was administered to a subgroup of stressed rats and found to be inactive against induction of CCL2, TNFα, or IL-1β mRNAs. These differential results suggest that stress and WCE manifest broad neuroimmune effects in brain depending on the cytokine and brain region, and that CRF inhibition may not be a relevant mechanism in non-alcohol exposed animals

Isolation and Characterization of Current Human Coronavirus Strains in Primary Human Epithelial Cell Cultures Reveal Differences in Target Cell Tropism

Science.gov (United States)

Dijkman, Ronald; Jebbink, Maarten F.; Koekkoek, Sylvie M.; Deijs, Martin; Jónsdóttir, Hulda R.; Molenkamp, Richard; Ieven, Margareta; Goossens, Herman; Thiel, Volker

2013-01-01

The human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n = 1), HCoV-NL63 (n = 1), HCoV-HKU1 (n = 4), and HCoV-OC43 (n = 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains. PMID:23427150

Proteomic analysis of purified coronavirus infectious bronchitis virus particles

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Shu Dingming

2010-06-01

Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

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Christine Burkard

2014-11-01

Full Text Available Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs. Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV. Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

Complete Genome Sequence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) from the First Imported MERS-CoV Case in China.

Science.gov (United States)

Lu, Roujian; Wang, Yanqun; Wang, Wenling; Nie, Kai; Zhao, Yanjie; Su, Juan; Deng, Yao; Zhou, Weimin; Li, Yang; Wang, Huijuan; Wang, Wen; Ke, Changwen; Ma, Xuejun; Wu, Guizhen; Tan, Wenjie

2015-08-13

On 26 May 2015, an imported Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in Guangdong Province, China, and found to be closely related to the MERS-CoV strain prevalent in South Korea. The full genome of the ChinaGD01 strain was sequenced and analyzed to investigate the epidemiology and evolution of MERS-CoV circulating in South Korea and China. Copyright © 2015 Lu et al.

A 3'-coterminal nested set of independently transcribed mRNAs is generated during Berne virus replication

International Nuclear Information System (INIS)

Snijder, E.J.; Horzinek, M.C.; Spaan, W.J.

1990-01-01

By using poly(A)-selected RNA from Berne virus (BEV)-infected embryonic mule skin cells as a template, cDNA was prepared and cloned in plasmid pUC9. Recombinants covering a contiguous sequence of about 10 kilobases were identified. Northern (RNA) blot hybridizations with various restriction fragments from these clones showed that the five BEV mRNAs formed a 3'-coterminal nested set. Sequence analysis revealed the presence of four complete open reading frames of 4743, 699, 426, and 480 nucleotides, with initiation codons coinciding with the 5' ends of BEV RNAs 2 through 5, respectively. By using primer extension analysis and oligonucleotide hybridizations, RNA 5 was found to be contiguous on the consensus sequence. The transcription of BEV mRNAs was studied by means of UV mapping. BEV RNAs 1, 2, and 3 were shown to be transcribed independently, which is also likely--although not rigorously proven--for RNAs 4 and 5. Upstream of the AUG codon of each open reading frame a conserved sequence pattern was observed which is postulated to function as a core promoter sequence in subgenomic RNA transcription. In the area surrounding the core promoter region of the two most abundant subgenomic BEV RNAs, a number of homologous sequence motifs were identified

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis

NARCIS (Netherlands)

Felten, Sandra; Leutenegger, Christian M.; Balzer, Hans Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman|info:eu-repo/dai/nl/089740890; Hartmann, Katrin

2017-01-01

Background: Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse

Detection of Avian coronavirus infectious bronchitis virus type QX infection in Switzerland.

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Sigrist, Brigitte; Tobler, Kurt; Schybli, Martina; Konrad, Leonie; Stöckli, René; Cattoli, Giovanni; Lüschow, Dörte; Hafez, Hafez M; Britton, Paul; Hoop, Richard K; Vögtlin, Andrea

2012-11-01

Infectious bronchitis, a disease of chickens caused by Avian coronavirus infectious bronchitis virus (IBV), leads to severe economic losses for the poultry industry worldwide. Various attempts to control the virus based on vaccination strategies are performed. However, due to the emergence of novel genotypes, an effective control of the virus is hindered. In 1996, a novel viral genotype named IBV-QX was reported for the first time in Qingdao, Shandong province, China. The first appearance of an IBV-QX isolate in Europe was reported between 2003 and 2004 in The Netherlands. Subsequently, infections with this genotype were found in several other European countries such as France, Italy, Germany, United Kingdom, Slovenia, and Sweden. The present report describes the use of a new set of degenerate primers that amplify a 636-bp fragment within the S1 gene by reverse transcription polymerase chain reaction to detect the occurrence of IBV-QX infection in Switzerland.

Feline coronavirus quantitative reverse transcriptase polymerase chain reaction on effusion samples in cats with and without feline infectious peritonitis.

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Longstaff, Louise; Porter, Emily; Crossley, Victoria J; Hayhow, Sophie E; Helps, Christopher R; Tasker, Séverine

2017-02-01

Objectives The aim of the study was to determine whether feline coronavirus (FCoV) RNA in effusion samples can be used as a diagnostic marker of feline infectious peritonitis (FIP); and in FCoV RNA-positive samples to examine amino acid codons in the FCoV spike protein at positions 1058 and 1060 where leucine and alanine, respectively, have been associated with systemic or virulent (FIP) FCoV infection. Methods Total RNA was extracted from effusion samples from 20 cats with confirmed FIP and 23 cats with other diseases. Feline coronavirus RNA was detected using a reverse transcriptase quantitative polymerase chain reaction assay (qRT-PCR), and positive samples underwent pyrosequencing of position 1058 with or without Sanger sequencing of position 1060 in the FCoV spike protein. Results Seventeen (85%) of the effusion samples from 20 cats with FIP were positive for FCoV RNA, whereas none of the 23 cats with other diseases were positive. Pyrosequencing of the 17 FCoV-positive samples showed that 11 (65%) of the cats had leucine and two (12%) had methionine at position 1058. Of the latter two samples with methionine, one had alanine at position 1060. Conclusions and relevance A positive FCoV qRT-PCR result on effusions appears specific for FIP and may be a useful diagnostic marker for FIP in cats with effusions. The majority of FCoVs contained amino acid changes previously associated with systemic spread or virulence (FIP) of the virus.

Organ-Specific and Age-Dependent Expression of Insulin-like Growth Factor-I (IGF-I) mRNA Variants: IGF-IA and IB mRNAs in the Mouse

OpenAIRE

Ohtsuki, Takashi; Otsuki, Mariko; Murakami, Yousuke; Maekawa, Tetsuya; Yamamoto, Takashi; Akasaka, Koji; Takeuchi, Sakae; Takahashi, Sumio

2005-01-01

Insulin-like growth factor-I (IGF-I) gene generates several IGF-I mRNA variants by alternative splicing. Two promoters are present in mouse IGF-I gene. Each promoter encodes two IGF-I mRNA variants (IGF-IA and IGF-IB mRNAs). Variants differ by the presence (IGF-IB) or absence (IGF-IA) of a 52-bp insert in the E domain-coding region. Functional differences among IGF-I mRNAs, and regulatory mechanisms for alternative splicing of IGF-I mRNA are not yet known. We analyzed the expression of mouse ...

Myeloablation-associated deletion of ORF4 in a human coronavirus 229E infection.

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Greninger, Alexander L; Pepper, Gregory; Shean, Ryan C; Cent, Anne; Palileo, Isabel; Kuypers, Jane M; Schiffer, Joshua T; Jerome, Keith R

2017-01-01

We describe metagenomic next-generation sequencing (mNGS) of a human coronavirus 229E from a patient with AML and persistent upper respiratory symptoms, who underwent hematopoietic cell transplantation (HCT). mNGS revealed a 548-nucleotide deletion, which comprised the near entirety of the ORF4 gene, and no minor allele variants were detected to suggest a mixed infection. As part of her pre-HCT conditioning regimen, the patient received myeloablative treatment with cyclophosphamide and 12 Gy total body irradiation. Iterative sequencing and RT-PCR confirmation of four respiratory samples over the 4-week peritransplant period revealed that the pre-conditioning strain contained an intact ORF4 gene, while the deletion strain appeared just after conditioning and persisted over a 2.5-week period. This sequence represents one of the largest genomic deletions detected in a human RNA virus and describes large-scale viral mutation associated with myeloablation for HCT.

Profiling and Co-expression Network Analysis of Learned Helplessness Regulated mRNAs and lncRNAs in the Mouse Hippocampus

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Chaoqun Li

2018-01-01

Full Text Available Although studies provide insights into the neurobiology of stress and depression, the exact molecular mechanisms underlying their pathologies remain largely unknown. Long non-coding RNA (lncRNA has been implicated in brain functions and behavior. A potential link between lncRNA and psychiatric disorders has been proposed. However, it remains undetermined whether IncRNA regulation, in the brain, contributes to stress or depression pathologies. In this study, we used a valid animal model of depression-like symptoms; namely learned helplessness, RNA-seq, Gene Ontology and co-expression network analyses to profile the expression pattern of lncRNA and mRNA in the hippocampus of mice. We identified 6346 differentially expressed transcripts. Among them, 340 lncRNAs and 3559 protein coding mRNAs were differentially expressed in helpless mice in comparison with control and/or non-helpless mice (inescapable stress resilient mice. Gene Ontology and pathway enrichment analyses indicated that induction of helplessness altered expression of mRNAs enriched in fundamental biological functions implicated in stress/depression neurobiology such as synaptic, metabolic, cell survival and proliferation, developmental and chromatin modification functions. To explore the possible regulatory roles of the altered lncRNAs, we constructed co-expression networks composed of the lncRNAs and mRNAs. Among our differentially expressed lncRNAs, 17% showed significant correlation with genes. Functional co-expression analysis linked the identified lncRNAs to several cellular mechanisms implicated in stress/depression neurobiology. Importantly, 57% of the identified regulatory lncRNAs significantly correlated with 18 different synapse-related functions. Thus, the current study identifies for the first time distinct groups of lncRNAs regulated by induction of learned helplessness in the mouse brain. Our results suggest that lncRNA-directed regulatory mechanisms might contribute to

Profiling and Co-expression Network Analysis of Learned Helplessness Regulated mRNAs and lncRNAs in the Mouse Hippocampus.

Science.gov (United States)

Li, Chaoqun; Cao, Feifei; Li, Shengli; Huang, Shenglin; Li, Wei; Abumaria, Nashat

2017-01-01

Although studies provide insights into the neurobiology of stress and depression, the exact molecular mechanisms underlying their pathologies remain largely unknown. Long non-coding RNA (lncRNA) has been implicated in brain functions and behavior. A potential link between lncRNA and psychiatric disorders has been proposed. However, it remains undetermined whether IncRNA regulation, in the brain, contributes to stress or depression pathologies. In this study, we used a valid animal model of depression-like symptoms; namely learned helplessness, RNA-seq, Gene Ontology and co-expression network analyses to profile the expression pattern of lncRNA and mRNA in the hippocampus of mice. We identified 6346 differentially expressed transcripts. Among them, 340 lncRNAs and 3559 protein coding mRNAs were differentially expressed in helpless mice in comparison with control and/or non-helpless mice (inescapable stress resilient mice). Gene Ontology and pathway enrichment analyses indicated that induction of helplessness altered expression of mRNAs enriched in fundamental biological functions implicated in stress/depression neurobiology such as synaptic, metabolic, cell survival and proliferation, developmental and chromatin modification functions. To explore the possible regulatory roles of the altered lncRNAs, we constructed co-expression networks composed of the lncRNAs and mRNAs. Among our differentially expressed lncRNAs, 17% showed significant correlation with genes. Functional co-expression analysis linked the identified lncRNAs to several cellular mechanisms implicated in stress/depression neurobiology. Importantly, 57% of the identified regulatory lncRNAs significantly correlated with 18 different synapse-related functions. Thus, the current study identifies for the first time distinct groups of lncRNAs regulated by induction of learned helplessness in the mouse brain. Our results suggest that lncRNA-directed regulatory mechanisms might contribute to stress

p53 down-regulates SARS coronavirus replication and is targeted by the SARS-unique domain and PLpro via E3 ubiquitin ligase RCHY1

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Ma-Lauer, Yue; Carbajo-Lozoya, Javier; Müller, Marcel A.; Deng, Wen; Lei, Jian; Meyer, Benjamin; Kusov, Yuri; von Brunn, Brigitte; Bairad, Dev Raj; Hünten, Sabine; Drosten, Christian; Hermeking, Heiko; Leonhardt, Heinrich; Mann, Matthias; Hilgenfeld, Rolf; von Brunn, Albrecht

2016-01-01

Highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) has developed strategies to inhibit host immune recognition. We identify cellular E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1) as an interacting partner of the viral SARS-unique domain (SUD) and papain-like protease (PLpro), and, as a consequence, the involvement of cellular p53 as antagonist of coronaviral replication. Residues 95–144 of RCHY1 and 389–652 of SUD (SUD-NM) subdomains are crucial for interaction. Association with SUD increases the stability of RCHY1 and augments RCHY1-mediated ubiquitination as well as degradation of p53. The calcium/calmodulin-dependent protein kinase II delta (CAMK2D), which normally influences RCHY1 stability by phosphorylation, also binds to SUD. In vivo phosphorylation shows that SUD does not regulate phosphorylation of RCHY1 via CAMK2D. Similarly to SUD, the PLpros from SARS-CoV, MERS-CoV, and HCoV-NL63 physically interact with and stabilize RCHY1, and thus trigger degradation of endogenous p53. The SARS-CoV papain-like protease is encoded next to SUD within nonstructural protein 3. A SUD–PLpro fusion interacts with RCHY1 more intensively and causes stronger p53 degradation than SARS-CoV PLpro alone. We show that p53 inhibits replication of infectious SARS-CoV as well as of replicons and human coronavirus NL63. Hence, human coronaviruses antagonize the viral inhibitor p53 via stabilizing RCHY1 and promoting RCHY1-mediated p53 degradation. SUD functions as an enhancer to strengthen interaction between RCHY1 and nonstructural protein 3, leading to a further increase in in p53 degradation. The significance of these findings is that down-regulation of p53 as a major player in antiviral innate immunity provides a long-sought explanation for delayed activities of respective genes. PMID:27519799

Conservation of nucleotide sequences for molecular diagnosis of Middle East respiratory syndrome coronavirus, 2015

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Yuki Furuse

2015-11-01

Full Text Available Infection due to the Middle East respiratory syndrome coronavirus (MERS-CoV is widespread. The present study was performed to assess the protocols used for the molecular diagnosis of MERS-CoV by analyzing the nucleotide sequences of viruses detected between 2012 and 2015, including sequences from the large outbreak in eastern Asia in 2015. Although the diagnostic protocols were established only 2 years ago, mismatches between the sequences of primers/probes and viruses were found for several of the assays. Such mismatches could lead to a lower sensitivity of the assay, thereby leading to false-negative diagnosis. A slight modification in the primer design is suggested. Protocols for the molecular diagnosis of viral infections should be reviewed regularly after they are established, particularly for viruses that pose a great threat to public health such as MERS-CoV.

Dromedary camels and the transmission of Middle East Respiratory Syndrome Coronavirus (MERS-CoV)

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Hemida, Maged G; Elmoslemany, Ahmed; Al-Hizab, Fahad; Alnaeem, Abdulmohsen; Almathen, Faisal; Faye, Bernard; Chu, Daniel KW; Perera, Ranawaka A; Peiris, Malik

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is an existential threat to global public health. The virus has been repeatedly detected in dromedary camels (Camelus dromedarius). Adult animals in many countries in the Middle East as well as in North and East Africa showed high (>90%) sero-prevalence to the virus. MERS-CoV isolated from dromedaries is genetically and phenotypically similar to viruses from humans. We summarise current understanding of the ecology of MERS-CoV in animals and transmission at the animal-human interface. We review aspects of husbandry, animal movements and trade and the use and consumption of camel dairy and meat products in the Middle East that may be relevant to the epidemiology of MERS. We also highlight the gaps in understanding the transmission of this virus in animals and from animals to humans. PMID:26256102

Detection of ascitic feline coronavirus RNA from cats with clinically suspected feline infectious peritonitis.

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Soma, Takehisa; Wada, Makoto; Taharaguchi, Satoshi; Tajima, Tomoko

2013-10-01

Ascitic feline coronavirus (FCoV) RNA was examined in 854 cats with suspected feline infectious peritonitis (FIP) by RT-PCR. The positivity was significantly higher in purebreds (62.2%) than in crossbreds (34.8%) (P<0.0001). Among purebreds, the positivities in the Norwegian forest cat (92.3%) and Scottish fold (77.6%) were significantly higher than the average of purebreds (P=0.0274 and 0.0251, respectively). The positivity was significantly higher in males (51.5%) than in females (35.7%) (P<0.0001), whereas no gender difference has generally been noted in FCoV antibody prevalence, indicating that FIP more frequently develops in males among FCoV-infected cats. Genotyping was performed for 377 gene-positive specimens. Type I (83.3%) was far more predominantly detected than type II (10.6%) (P<0.0001), similar to previous serological and genetic surveys.

Sero-prevalence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV) specific antibodies in Dromedary Camels in Tabuk, Saudi Arabia.

Science.gov (United States)

Harrath, Rafik; Duhier, Faisel M Abu

2018-04-16

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a novel Coronavirus which was responsible of the first case of human acute respiratory syndrome in the Kingdom of Saudi Arabia (KSA), 2012. Dromedary camels are considered as potential reservoirs for the virus and seem to be the only animal host which may transmit the infection to human. Further studies are required to better understand the animal sources of zoonotic transmission route and the risks of this infection. A primary sero-prevalence study of MERS-CoV preexisting neutralizing antibodies in Dromedary camel serum was conducted in Tabuk, western north region of KSA, in order to assess the seopositivity of these animals and to explain their possible role in the transmission of the infection to Human. One hundred seventy one (171) serum samples were collected from healthy dromedary camels with different ages and genders in Tabuk city and tested for specific serum IgG by ELISA using the receptor-binding S1 subunits of spike proteins of MERS-CoV. 144 (84,21%) of the total camel sera shown the presence of protein-specific antibodies against MERS-CoV. These results may provide evidence that MERS-CoV has previously infected dromedary camels in Tabuk and may support the possible role of camels in the human infection. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Differential stability of host mRNAs in Friend erythroleukemia cells infected with herpes simplex virus type 1

International Nuclear Information System (INIS)

Mayman, B.A.; Nishioka, Y.

1985-01-01

The consequences of herpes simplex virus type 1 infection on cellular macromolecules were investigated in Friend erythroleukemia cells. The patterns of protein synthesis, examined by polyacrylamide gel electrophoresis, demonstrated that by 4 h postinfection the synthesis of many host proteins, with the exception of histones, was inhibited. Examination of the steady-state level of histone H3 mRNA by molecular hybridization of total RNA to a cloned mouse histone H3 complementary DNA probe demonstrated that the ratio of histone H3 mRNA to total RNA remained unchanged for the first 4 h postinfection. In contrast, the steady-state levels of globin and actin mRNAs decreased progressively at early intervals postinfection. Studies on RNA synthesis in isolated nuclei demonstrated that the transcription of the histone H3 gene was inhibited to approximately the same extent as that of actin gene. It was concluded that the stabilization of preexisting histone H3 mRNA was responsible for the persistence of H3 mRNA and histone protein synthesis in herpes simplex virus type 1-infected Friend erythroleukemia cells. The possible mechanisms influencing the differential stability of host mRNAs during the course of productive infection with herpes simplex virus type 1 are discussed

Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein

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Millet, Jean Kaoru; Whittaker, Gary R.

2014-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2′ position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2′ site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus. PMID:25288733

A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

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Wu, Kailang; Chen, Lang; Peng, Guiqing; Zhou, Wenbo; Pennell, Christopher A; Mansky, Louis M; Geraghty, Robert J; Li, Fang

2011-06-01

How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

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Chen Jing

2010-04-01

Full Text Available Abstract Background The discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs. Methods The ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip, provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established. Results We used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+ and MDA-MB-231 (estrogen receptor negative, ER- breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis. Conclusion This is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular

Neuroblastoma mRNAs predict outcome in children with stage 4 neuroblastoma: a European HR-NBL1/SIOPEN study.

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Viprey, Virginie F; Gregory, Walter M; Corrias, Maria V; Tchirkov, Andrei; Swerts, Katrien; Vicha, Ales; Dallorso, Sandro; Brock, Penelope; Luksch, Roberto; Valteau-Couanet, Dominique; Papadakis, Vassilios; Laureys, Genevieve; Pearson, Andrew D; Ladenstein, Ruth; Burchill, Susan A

2014-04-01

To evaluate the hypothesis that detection of neuroblastoma mRNAs by reverse transcriptase quantitative polymerase chain reaction (RTqPCR) in peripheral blood (PB) and bone marrow aspirates (BM) from children with stage 4 neuroblastoma are clinically useful biomarkers of risk. RTqPCR for paired-like homeobox 2b (PHOX2B), tyrosine hydroxylase (TH), and doublecortin (DCX) mRNA in PB and BM of children enrolled onto the High-Risk Neuroblastoma Trial-1 of the European Society of Pediatric Oncology Neuroblastoma Group (HR-NBL1/SIOPEN) was performed at diagnosis and after induction therapy. High levels of TH, PHOX2B, or DCX mRNA in PB or BM at diagnosis strongly predicted for worse event-free survival (EFS) and overall survival (OS) in a cohort of 290 children. After induction therapy, high levels of these mRNAs predicted worse EFS and OS in BM but not in PB. Combinations of mRNAs in BM did not add to the predictive power of any single mRNA. However, in the original (n = 182) and validation (n = 137) PB cohorts, high TH (log10TH > 0.8) or high PHOX2B (log10PHOX2B > 0.28) identify 19% of children as ultrahigh risk, with 5-year EFS and OS rates of 0%; OS rate was 25% (95% CI, 16% to 36%) and EFS rate was 38% (95% CI, 28% to 49%) in the remaining children. The magnitude of reduction in mRNA level between diagnosis and postinduction therapy in BM or PB was not of additional predictive value. High levels of TH and PHOX2B mRNA in PB at diagnosis objectively identify children with ultrahigh-risk disease who may benefit from novel treatment approaches. The level of TH, PHOX2B, and DCX mRNA in BM and/or PB at diagnosis might contribute to an algorithm to improve stratification of children for treatment.

Molecular characterization of feline infectious peritonitis virus strain DF-2 and studies of the role of ORF3abc in viral cell tropism.

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Bálint, Ádám; Farsang, Attila; Zádori, Zoltán; Hornyák, Ákos; Dencso, László; Almazán, Fernando; Enjuanes, Luis; Belák, Sándor

2012-06-01

The full-length genome of the highly lethal feline infectious peritonitis virus (FIPV) strain DF-2 was sequenced and cloned into a bacterial artificial chromosome (BAC) to study the role of ORF3abc in the FIPV-feline enteric coronavirus (FECV) transition. The reverse genetic system allowed the replacement of the truncated ORF3abc of the original FIPV DF-2 genome with the intact ORF3abc of the canine coronavirus (CCoV) reference strain Elmo/02. The in vitro replication kinetics of these two viruses was studied in CrFK and FCWF-4 cell lines, as well as in feline peripheral blood monocytes. Both viruses showed similar replication kinetics in established cell lines. However, the strain with a full-length ORF3 showed markedly lower replication of more than 2 log(10) titers in feline peripheral blood monocytes. Our results suggest that the truncated ORF3abc plays an important role in the efficient macrophage/monocyte tropism of type II FIPV.

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

International Nuclear Information System (INIS)

Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong

2012-01-01

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

Antibodies against MERS coronavirus in dromedary camels, United Arab Emirates, 2003 and 2013.

Science.gov (United States)

Meyer, Benjamin; Müller, Marcel A; Corman, Victor M; Reusken, Chantal B E M; Ritz, Daniel; Godeke, Gert-Jan; Lattwein, Erik; Kallies, Stephan; Siemens, Artem; van Beek, Janko; Drexler, Jan F; Muth, Doreen; Bosch, Berend-Jan; Wernery, Ulrich; Koopmans, Marion P G; Wernery, Renate; Drosten, Christian

2014-04-01

Middle East respiratory syndrome coronavirus (MERS-CoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedary camels have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary camel serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) camels had antibodies against MERS-CoV. This result included all 151 serum samples obtained in 2003. Most (389/651, 59.8%) serum samples had MERS-CoV-neutralizing antibody titers >1,280. Dromedary camels from the United Arab Emirates were infected at high rates with MERS-CoV or a closely related, probably conspecific, virus long before the first human MERS cases.

Replication of murine coronavirus requires multiple cysteines in the endodomain of spike protein

Energy Technology Data Exchange (ETDEWEB)

Yang, Jinhua; Lv, Jun; Wang, Yuyan; Gao, Shuang; Yao, Qianqian; Qu, Di; Ye, Rong, E-mail: yerong24@fudan.edu.cn

2012-06-05

A conserved cysteine-rich motif located between the transmembrane domain and the endodomain is essential for membrane fusion and assembly of coronavirus spike (S) protein. Here, we proved that three cysteines within the motif, but not dependent on position, are minimally required for the survival of the recombinant mouse hepatitis virus. When the carboxy termini with these mutated motifs of S proteins were respectively introduced into a heterogeneous protein, both incorporation into lipid rafts and S-palmitoylation of these recombinant proteins showed a similar quantity requirement to cysteine residues. Meanwhile, the redistribution of these proteins on cellular surface indicated that the absence of the positively charged rather than cysteine residues in the motif might lead the dramatic reduction in syncytial formation of some mutants with the deleted motifs. These results suggest that multiple cysteine as well as charged residues concurrently improves the membrane-associated functions of S protein in viral replication and cytopathogenesis.

Drosophila Pumilio protein contains multiple autonomous repression domains that regulate mRNAs independently of Nanos and brain tumor.

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Weidmann, Chase A; Goldstrohm, Aaron C

2012-01-01

Drosophila melanogaster Pumilio is an RNA-binding protein that potently represses specific mRNAs. In developing embryos, Pumilio regulates a key morphogen, Hunchback, in collaboration with the cofactor Nanos. To investigate repression by Pumilio and Nanos, we created cell-based assays and found that Pumilio inhibits translation and enhances mRNA decay independent of Nanos. Nanos robustly stimulates repression through interactions with the Pumilio RNA-binding domain. We programmed Pumilio to recognize a new binding site, which garners repression of new target mRNAs. We show that cofactors Brain Tumor and eIF4E Homologous Protein are not obligatory for Pumilio and Nanos activity. The conserved RNA-binding domain of Pumilio was thought to be sufficient for its function. Instead, we demonstrate that three unique domains in the N terminus of Pumilio possess the major repressive activity and can function autonomously. The N termini of insect and vertebrate Pumilio and Fem-3 binding factors (PUFs) are related, and we show that corresponding regions of human PUM1 and PUM2 have repressive activity. Other PUF proteins lack these repression domains. Our findings suggest that PUF proteins have evolved new regulatory functions through protein sequences appended to their conserved PUF repeat RNA-binding domains.

Induction of Apoptosis by the Severe Acute Respiratory Syndrome Coronavirus 7a Protein Is Dependent on Its Interaction with the Bcl-XL Protein▿

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Tan, Ying-Xim; Tan, Timothy H. P.; Lee, Marvin J.-R.; Tham, Puay-Yoke; Gunalan, Vithiagaran; Druce, Julian; Birch, Chris; Catton, Mike; Fu, Nai Yang; Yu, Victor C.; Tan, Yee-Joo

2007-01-01

The severe acute respiratory syndrome coronavirus (SARS-CoV) 7a protein, which is not expressed by other known coronaviruses, can induce apoptosis in various cell lines. In this study, we show that the overexpression of Bcl-XL, a prosurvival member of the Bcl-2 family, blocks 7a-induced apoptosis, suggesting that the mechanism for apoptosis induction by 7a is at the level of or upstream from the Bcl-2 family. Coimmunoprecipitation experiments showed that 7a interacts with Bcl-XL and other prosurvival proteins (Bcl-2, Bcl-w, Mcl-1, and A1) but not with the proapoptotic proteins (Bax, Bak, Bad, and Bid). A good correlation between the abilities of 7a deletion mutants to induce apoptosis and to interact with Bcl-XL was observed, suggesting that 7a triggers apoptosis by interfering directly with the prosurvival function of Bcl-XL. Interestingly, amino acids 224 and 225 within the C-terminal transmembrane domain of Bcl-XL are essential for the interaction with the 7a protein, although the BH3 domain of Bcl-XL also contributes to this interaction. In addition, fractionation experiments showed that 7a colocalized with Bcl-XL at the endoplasmic reticulum as well as the mitochondria, suggesting that they may form complexes in different membranous compartments. PMID:17428862

Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

Science.gov (United States)

Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

2013-12-01

Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

Structural Analysis of Major Species Barriers between Humans and Palm Civets for Severe Acute Respiratory Syndrome Coronavirus Infections

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Li, Fang (UMM)

2008-09-23

It is believed that a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), was passed from palm civets to humans and caused the epidemic of SARS in 2002 to 2003. The major species barriers between humans and civets for SARS-CoV infections are the specific interactions between a defined receptor-binding domain (RBD) on a viral spike protein and its host receptor, angiotensin-converting enzyme 2 (ACE2). In this study a chimeric ACE2 bearing the critical N-terminal helix from civet and the remaining peptidase domain from human was constructed, and it was shown that this construct has the same receptor activity as civet ACE2. In addition, crystal structures of the chimeric ACE2 complexed with RBDs from various human and civet SARS-CoV strains were determined. These structures, combined with a previously determined structure of human ACE2 complexed with the RBD from a human SARS-CoV strain, have revealed a structural basis for understanding the major species barriers between humans and civets for SARS-CoV infections. They show that the major species barriers are determined by interactions between four ACE2 residues (residues 31, 35, 38, and 353) and two RBD residues (residues 479 and 487), that early civet SARS-CoV isolates were prevented from infecting human cells due to imbalanced salt bridges at the hydrophobic virus/receptor interface, and that SARS-CoV has evolved to gain sustained infectivity for human cells by eliminating unfavorable free charges at the interface through stepwise mutations at positions 479 and 487. These results enhance our understanding of host adaptations and cross-species infections of SARS-CoV and other emerging animal viruses.

Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

Science.gov (United States)

Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

2017-06-15

Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

Detection and genetic characterization of Canine parvovirus and Canine coronavirus strains circulating in district of Tirana in Albania.

Science.gov (United States)

Cavalli, Alessandra; Desario, Costantina; Kusi, Ilir; Mari, Viviana; Lorusso, Eleonora; Cirone, Francesco; Kumbe, Ilirjan; Colaianni, Maria Loredana; Buonavoglia, Domenico; Decaro, Nicola

2014-07-01

An epidemiological survey for Canine parvovirus 2 (CPV-2) and Canine coronavirus (CCoV) was conducted in Albania. A total of 57 fecal samples were collected from diarrheic dogs in the District of Tirana during 2011-2013. The molecular assays detected 53 and 31 CPV- and CCoV-positive specimens, respectively, with mixed CPV-CCoV infections diagnosed in 28 dogs. The most frequently detected CPV type was 2a, whereas IIa was the predominant CCoV subtype. A better comprehension of the CPV-CCoV epidemiology in eastern European countries will help to assess the most appropriate vaccination strategies to prevent disease due to infections with these widespread agents of acute gastroenteritis in the dog.

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro

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Jin Soo Shin

2018-05-01

Full Text Available The Middle East respiratory syndrome-coronavirus (MERS-CoV, first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK, was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

Saracatinib Inhibits Middle East Respiratory Syndrome-Coronavirus Replication In Vitro.

Science.gov (United States)

Shin, Jin Soo; Jung, Eunhye; Kim, Meehyein; Baric, Ralph S; Go, Yun Young

2018-05-24

The Middle East respiratory syndrome-coronavirus (MERS-CoV), first identified in Saudi Arabia, is an emerging zoonotic pathogen that causes severe acute respiratory illness in humans with a high fatality rate. Since its emergence, MERS-CoV continues to spread to countries outside of the Arabian Peninsula and gives rise to sporadic human infections following the entry of infected individuals to other countries, which can precipitate outbreaks similar to the one that occurred in South Korea in 2015. Current therapeutics against MERS-CoV infection have primarily been adapted from previous drugs used for the treatment of severe acute respiratory syndrome. In search of new potential drug candidates, we screened a library composed of 2334 clinically approved drugs and pharmacologically active compounds. The drug saracatinib, a potent inhibitor of Src-family of tyrosine kinases (SFK), was identified as an inhibitor of MERS-CoV replication in vitro. Our results suggest that saracatinib potently inhibits MERS-CoV at the early stages of the viral life cycle in Huh-7 cells, possibly through the suppression of SFK signaling pathways. Furthermore, saracatinib exhibited a synergistic effect with gemcitabine, an anticancer drug with antiviral activity against several RNA viruses. These data indicate that saracatinib alone or in combination with gemcitabine can provide a new therapeutic option for the treatment of MERS-CoV infection.

Deadenylase depletion protects inherited mRNAs in primordial germ cells.

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Swartz, S Zachary; Reich, Adrian M; Oulhen, Nathalie; Raz, Tal; Milos, Patrice M; Campanale, Joseph P; Hamdoun, Amro; Wessel, Gary M

2014-08-01

A crucial event in animal development is the specification of primordial germ cells (PGCs), which become the stem cells that create sperm and eggs. How PGCs are created provides a valuable paradigm for understanding stem cells in general. We find that the PGCs of the sea urchin Strongylocentrotus purpuratus exhibit broad transcriptional repression, yet enrichment for a set of inherited mRNAs. Enrichment of several germline determinants in the PGCs requires the RNA-binding protein Nanos to target the transcript that encodes CNOT6, a deadenylase, for degradation in the PGCs, thereby creating a stable environment for RNA. Misexpression of CNOT6 in the PGCs results in their failure to retain Seawi transcripts and Vasa protein. Conversely, broad knockdown of CNOT6 expands the domain of Seawi RNA as well as exogenous reporters. Thus, Nanos-dependent spatially restricted CNOT6 differential expression is used to selectively localize germline RNAs to the PGCs. Our findings support a 'time capsule' model of germline determination, whereby the PGCs are insulated from differentiation by retaining the molecular characteristics of the totipotent egg and early embryo. © 2014. Published by The Company of Biologists Ltd.

Excretion and detection of SARS coronavirus and its nucleic acid from digestive system

Science.gov (United States)

Wang, Xin-Wei; Li, Jin-Song; Guo, Ting-Kai; Zhen, Bei; Kong, Qing-Xin; Yi, Bin; Li, Zhong; Song, Nong; Jin, Min; Wu, Xiao-Ming; Xiao, Wen-Jun; Zhu, Xiu-Mei; Gu, Chang-Qing; Yin, Jing; Wei, Wei; Yao, Wei; Liu, Chao; Li, Jian-Feng; Ou, Guo-Rong; Wang, Min-Nian; Fang, Tong-Yu; Wang, Gui-Jie; Qiu, Yao-Hui; Wu, Huai-Huan; Chao, Fu-Huan; Li, Jun-Wen

2005-01-01

AIM: To study whether severe acute respiratory syndrome coronavirus (SARS-CoV) could be excreted from digestive system. METHODS: Cell culture and semi-nested RT-PCR were used to detect SARS-CoV and its RNA from 21 stool and urine samples, and a kind of electropositive filter media particles was used to concentrate the virus in 10 sewage samples from two hospitals receiving SARS patients in Beijing in China. RESULTS: It was demonstrated that there was no live SARS-CoV in all samples collected, but the RNA of SARS-CoV could be detected in seven stool samples from SARS patients with any one of the symptoms of fever, malaise, cough, or dyspnea, in 10 sewage samples before disinfection and 3 samples after disinfection from the two hospitals. The RNA could not be detected in urine and stool samples from patients recovered from SARS. CONCLUSION: Nucleic acid of SARS-CoV can be excreted through the stool of patients into sewage system, and the possibility of SARS-CoV transmitting through digestive system cannot be excluded. PMID:16038039

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

International Nuclear Information System (INIS)

Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre

2006-01-01

Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4 1 32 or P4 3 32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å

Severe respiratory illness associated with a novel coronavirus--Saudi Arabia and Qatar, 2012.

Science.gov (United States)

2012-10-12

CDC is working closely with the World Health Organization (WHO) and other partners to better understand the public health risk presented by a recently detected, novel coronavirus. This virus has been identified in two patients, both previously healthy adults who suffered severe respiratory illness. The first patient, a man aged 60 years from Saudi Arabia, was hospitalized in June 2012 and died; the second patient, a man aged 49 years from Qatar with onset of symptoms in September 2012 was transported to the United Kingdom for intensive care. He remains hospitalized on life support with both pulmonary and renal failure. Person-to-person or health-care-associated transmission has not been identified to date. Interim case definitions based on acute respiratory illness and travel history were issued by WHO on September 29 and include criteria for "patient under investigation," "probable case," and "confirmed case". This information is current as of October 4. Updates on the investigation and the WHO case definition are available at http://www.who.int/csr/don/en/index.html.

Protein-Protein Interactions of Viroporins in Coronaviruses and Paramyxoviruses: New Targets for Antivirals?

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Jaume Torres

2015-06-01

Full Text Available Viroporins are members of a rapidly growing family of channel-forming small polypeptides found in viruses. The present review will be focused on recent structural and protein-protein interaction information involving two viroporins found in enveloped viruses that target the respiratory tract; (i the envelope protein in coronaviruses and (ii the small hydrophobic protein in paramyxoviruses. Deletion of these two viroporins leads to viral attenuation in vivo, whereas data from cell culture shows involvement in the regulation of stress and inflammation. The channel activity and structure of some representative members of these viroporins have been recently characterized in some detail. In addition, searches for protein-protein interactions using yeast-two hybrid techniques have shed light on possible functional roles for their exposed cytoplasmic domains. A deeper analysis of these interactions should not only provide a more complete overview of the multiple functions of these viroporins, but also suggest novel strategies that target protein-protein interactions as much needed antivirals. These should complement current efforts to block viroporin channel activity.

Crystallization and preliminary X-ray diffraction analysis of Nsp15 from SARS coronavirus

Energy Technology Data Exchange (ETDEWEB)

Ricagno, Stéfano; Coutard, Bruno; Grisel, Sacha; Brémond, Nicolas; Dalle, Karen; Tocque, Fabienne; Campanacci, Valérie; Lichière, Julie; Lantez, Violaine; Debarnot, Claire; Cambillau, Christian; Canard, Bruno; Egloff, Marie-Pierre, E-mail: marie-pierre.egloff@afmb.univ-mrs.fr [Centre National de la Recherche Scientifique and Universités d’Aix-Marseille I et II, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Ecole Supérieure d’Ingénieurs de Luminy-Case 925, 163 Avenue de Luminy, 13288 Marseille CEDEX 9 (France)

2006-04-01

Crystals of Nsp15 from the aetiological agent of SARS have been grown at room temperature. Crystals have cubic symmetry and diffract to a maximum resolution of 2.7 Å. The non-structural protein Nsp15 from the aetiological agent of SARS (severe acute respiratory syndrome) has recently been characterized as a uridine-specific endoribonuclease. This enzyme plays an essential role in viral replication and transcription since a mutation in the related H229E human coronavirus nsp15 gene can abolish viral RNA synthesis. SARS full-length Nsp15 (346 amino acids) has been cloned and expressed in Escherichia coli with an N-terminal hexahistidine tag and has been purified to homogeneity. The protein was subsequently crystallized using PEG 8000 or 10 000 as precipitants. Small cubic crystals of the apoenzyme were obtained from 100 nl nanodrops. They belong to space group P4{sub 1}32 or P4{sub 3}32, with unit-cell parameters a = b = c = 166.8 Å. Diffraction data were collected to a maximum resolution of 2.7 Å.

Characterization of an Immunodominant Epitope in the Endodomain of the Coronavirus Membrane Protein

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Hui Dong

2016-12-01

Full Text Available The coronavirus membrane (M protein acts as a dominant immunogen and is a major player in virus assembly. In this study, we prepared two monoclonal antibodies (mAbs; 1C3 and 4C7 directed against the transmissible gastroenteritis virus (TGEV M protein. The 1C3 and 4C7 mAbs both reacted with the native TGEV M protein in western blotting and immunofluorescence (IFA assays. Two linear epitopes, 243YSTEART249 (1C3 and 243YSTEARTDNLSEQEKLLHMV262 (4C7, were identified in the endodomain of the TGEV M protein. The 1C3 mAb can be used for the detection of the TGEV M protein in different assays. An IFA method for the detection of TGEV M protein was optimized using mAb 1C3. Furthermore, the ability of the epitope identified in this study to stimulate antibody production was also evaluated. An immunodominant epitope in the TGEV membrane protein endodomain was identified. The results of this study have implications for further research on TGEV replication.

Characterization and distribution of GHRH, PACAP, TRH, SST and IGF1 mRNAs in the green iguana.

Science.gov (United States)

Ávila-Mendoza, José; Pérez-Rueda, Ernesto; Urban-Sosa, Valeria; Carranza, Martha; Martínez-Moreno, Carlos G; Luna, Maricela; Arámburo, Carlos

2018-01-01

The somatotropic axis (SA) regulates numerous aspects of vertebrate physiology such as development, growth, and metabolism and has influence on several tissues including neural, immune, reproductive and gastric tract. Growth hormone (GH) is a key component of SA, it is synthesized and released mainly by pituitary somatotrophs, although now it is known that virtually all tissues can express GH, which, in addition to its well-described endocrine roles, also has autocrine/paracrine/intracrine actions. In the pituitary, GH expression is regulated by several hypothalamic neuropeptides including GHRH, PACAP, TRH and SST. GH, in turn, regulates IGF1 synthesis in several target tissues, adding complexity to the system since GH effects can be exerted either directly or mediated by IGF1. In reptiles, little is known about the SA components and their functional interactions. The aim of this work was to characterize the mRNAs of the principal SA components in the green iguana and to develop the tools that allow the study of the structural and functional evolution of this system in reptiles. By employing RT-PCR and RACE, the cDNAs encoding for GHRH, PACAP, TRH, SST and IGF1 were amplified and sequenced. Results showed that these cDNAs coded for the corresponding protein precursors of 154, 170, 243, 113, and 131 amino acids, respectively. Of these, GHRH, PACAP, SST and IGF1 precursors exhibited a high structural conservation with respect to its counterparts in other vertebrates. On the other hand, iguana's TRH precursor showed 7 functional copies of mature TRH (pyr-QHP-NH 2 ), as compared to 4 and 6 copies of TRH in avian and mammalian proTRH sequences, respectively. It was found that in addition to its primary production site (brain for GHRH, PACAP, TRH and SST, and liver for IGF1), they were also expressed in other peripheral tissues, i.e. testes and ovaries expressed all the studied mRNAs, whereas TRH and IGF1 mRNAs were observed ubiquitously in all tissues considered. These

A comparative kinetic RT/-PCR strategy for the quantitation of mRNAs in microdissected human renal biopsy specimens.

Science.gov (United States)

Del Prete, D; Forino, M; Gambaro, G; D'Angelo, A; Baggio, B; Anglani, F

1998-01-01

Molecular biology techniques, to be applicable to a diagnostic renal biopsy specimen, should (1) be highly sensitive to be performed on a very small quantity of tissue; (2) be quantitative because they have to analyze genes normally expressed in the tissue and (3) allow the analysis of as large a number of genes as possible. Among different methods, only the reverse-transcriptase polymerase chain reaction (RT/-PCR) might comply with previous requisites, but the few RT/-PCR examples on renal biopsies in the literature do not allow starting RNA quantification and quality control; furthermore they have the drawback of analyzing only few genes. In an ongoing study to assess the expression of a number of genes in glomeruli and in tubulointerstitium of patients with different nephropathies, we developed a comparative RT/-PCR kinetic strategy based on the purification and quantification of total glomerular and tubulointerstitial RNA and on the use of an internal standard, the housekeeping gene G3PDH. We demonstrate that in microdissected diagnostic renal biopsies (1) glomerular and interstitial starting RNA can be quantified; (2) the G3PDH gene may be used both as an internal standard and as an indirect marker of RNA integrity; (3) as low as 28 ng of total RNA is sufficient to obtain PCR products of eight genes, and (4) it is worth to operate on microdissected biopsy specimens because of the different expression of genes in the two renal compartments.

Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

International Nuclear Information System (INIS)

Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

1987-01-01

Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA 1 ) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [ 35 S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs

Highly efficient gene knockout by injection of TALEN mRNAs into oocytes and host transfer in Xenopus laevis

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Keisuke Nakajima

2015-01-01

Full Text Available Zinc-finger nucleases, transcription activator-like effector nucleases (TALENs and the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins system are potentially powerful tools for producing tailor-made knockout animals. However, their mutagenic activity is not high enough to induce mutations at all loci of a target gene throughout an entire tadpole. In this study, we present a highly efficient method for introducing gene modifications at almost all target sequences in randomly selected embryos. The gene modification activity of TALEN is enhanced by adopting the host-transfer technique. In our method, the efficiency is further improved by injecting TALEN mRNAs fused to the 3′UTR of the Xenopus DEADSouth gene into oocytes, which are then transferred into a host female frog, where they are ovulated and fertilized. The addition of the 3′UTR of the DEADSouth gene promotes mRNA translation in the oocytes and increases the expression of TALEN proteins to near-maximal levels three hours post fertilization (hpf. In contrast, TALEN mRNAs without this 3′UTR are translated infrequently in oocytes. Our data suggest that genomic DNA is more sensitive to TALEN proteins from fertilization to the midblastula (MBT stage. Our method works by increasing the levels of TALEN proteins during the pre-MBT stages.

Experimental inoculation of equine coronavirus into Japanese draft horses.

Science.gov (United States)

Nemoto, Manabu; Oue, Yasuhiro; Morita, Yoshinori; Kanno, Toru; Kinoshita, Yuta; Niwa, Hidekazu; Ueno, Takanori; Katayama, Yoshinari; Bannai, Hiroshi; Tsujimura, Koji; Yamanaka, Takashi; Kondo, Takashi

2014-12-01

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

Prevalence of comorbidities in the Middle East respiratory syndrome coronavirus (MERS-CoV: a systematic review and meta-analysis

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Alaa Badawi

2016-08-01

Full Text Available The Middle East respiratory syndrome coronavirus (MERS-CoV is associated with life-threatening severe illnesses and a mortality rate of approximately 35%, particularly in patients with underlying comorbidities. A systematic analysis of 637 MERS-CoV cases suggests that diabetes and hypertension are equally prevalent in approximately 50% of the patients. Cardiac diseases are present in 30% and obesity in 16% of the cases. These conditions down-regulate the synthesis of proinflammatory cytokines and impair the host's innate and humoral immune systems. In conclusion, protection against MERS-CoV and other respiratory infections can be improved if public health vaccination strategies are tailored to target persons with chronic disorders.

Full-length genome sequences of porcine epidemic diarrhoea virus strain CV777; Use of NGS to analyse genomic and sub-genomic RNAs

DEFF Research Database (Denmark)

Rasmussen, Thomas Bruun; Boniotti, Maria Beatrice; Papetti, Alice

2018-01-01

Porcine epidemic diarrhoea virus, strain CV777, was initially characterized in 1978 as the causative agent of a disease first identified in the UK in 1971. This coronavirus has been widely distributed among laboratories and has been passaged both within pigs and in cell culture. To determine...... the variability between different stocks of the PEDV strain CV777, sequencing of the full-length genome (ca. 28kb) has been performed in 6 different laboratories, using different protocols. Not surprisingly, each of the different full genome sequences were distinct from each other and from the reference sequence...... the analysis of sub-genomic mRNAs from infected cells. It is clearly important to know the features of the specific sample of CV777 being used for experimental studies....

An "up, no change, or down" system: Time-dependent expression of mRNAs in contused skeletal muscle of rats used for wound age estimation.

Science.gov (United States)

Sun, Jun-Hong; Zhu, Xi-Yan; Dong, Ta-Na; Zhang, Xiao-Hong; Liu, Qi-Qing; Li, San-Qiang; Du, Qiu-Xiang

2017-03-01

The combined use of multiple markers is considered a promising strategy in estimating the age of wounds. We sought to develop an "up, no change, or down" system and to explore how to combine and use various parameters. In total, 78 Sprague Dawley rats were divided randomly into a control group and contusion groups of 4-, 8-, 12-, 16-, 20-, 24-, 28-, 32-, 36-, 40-, 44-, and 48-h post-injury (n=6 per group). A contusion was produced in the right limb of the rats under diethyl ether anesthesia by a drop-ball technique; the animals were sacrificed at certain time points thereafter, using a lethal dose of pentobarbital. Levels of PUM2, TAB2, GJC1, and CHRNA1 mRNAs were detected in contused muscle using real-time PCR. An up, no change, or down system was developed with the relative quantities of the four mRNAs recorded as black, dark gray, or light gray boxes, representing up-, no change, or down-regulation of the gene of interest during wound repair. The four transcripts were combined and used as a marker cluster for color model analysis of each contusion group. Levels of PUM2, TAB2, and GJC1 mRNAs decreased, whereas that of CHRNA1 increased in wound repair (Psystem was adequate to distinguish most time groups with the color model. Thus, the proposed up, no change, or down system provide the means to determine the minimal periods of early wounds. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Middle East respiratory syndrome coronavirus specific antibodies in naturally exposed Israeli llamas, alpacas and camels

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Dan David

2018-06-01

Full Text Available Thus far, no human MERS-CoV infections have been reported from Israel. Evidence for the circulation of MERS-CoV in dromedaries has been reported from almost all the countries of the Middle East, except Israel. Therefore, we aimed to analyze MERS-CoV infection in Israeli camelids, sampled between 2012 and 2017. A total of 411 camels, 102 alpacas and 19 llamas' sera were tested for the presence of antibodies to MERS-CoV. Our findings indicate a lower MERS-CoV seropositivity among Israeli dromedaries than in the surrounding countries, and for the first time naturally infected llamas were identified. In addition, nasal swabs of 661 camels, alpacas and lamas, obtained from January 2015 to December 2017, were tested for the presence of MERS-CoV RNA. All nasal swabs were negative, indicating no evidence for MERS-CoV active circulation in these camelids during that time period. Keywords: MERS coronavirus, Antibodies, Israel, Dromedary camels, Llamas, Alpacas

A phylogenetically distinct Middle East respiratory syndrome coronavirus detected in a dromedary calf from a closed dairy herd in Dubai with rising seroprevalence with age

OpenAIRE

Wernery, Ulrich; Rasoul, IHassab El; Wong, Emily YM; Joseph, Marina; Chen, Yixin; Jose, Shanty; Tsang, Alan KL; Patteril, Nissy Annie Georgy; Chen, Honglin; Elizabeth, Shyna K; Yuen, Kwok-Yung; Joseph, Sunitha; Xia, Ningshao; Wernery, Renate; Lau, Susanna KP

2015-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) was detected by monoclonal antibody-based nucleocapsid protein-capture enzyme-linked immunosorbent assay (ELISA), RNA detection, and viral culture from the nasal sample of a 1-month-old dromedary calf in Dubai with sudden death. Whole genome phylogeny showed that this MERS-CoV strain did not cluster with the other MERS-CoV strains from Dubai that we reported recently. Instead, it formed a unique branch more closely related to other MERS-...

Prolonged Shedding of Human Coronavirus in Hematopoietic Cell Transplant Recipients: Risk Factors and Viral Genome Evolution.

Science.gov (United States)

Ogimi, Chikara; Greninger, Alexander L; Waghmare, Alpana A; Kuypers, Jane M; Shean, Ryan C; Xie, Hu; Leisenring, Wendy M; Stevens-Ayers, Terry L; Jerome, Keith R; Englund, Janet A; Boeckh, Michael

2017-07-15

Recent data suggest that human coronavirus (HCoV) pneumonia is associated with significant mortality in hematopoietic cell transplant (HCT) recipients. Investigation of risk factors for prolonged shedding and intrahost genome evolution may provide critical information for development of novel therapeutics. We retrospectively reviewed HCT recipients with HCoV detected in nasal samples by polymerase chain reaction (PCR). HCoV strains were identified using strain-specific PCR. Shedding duration was defined as time between first positive and first negative sample. Logistic regression analyses were performed to evaluate factors for prolonged shedding (≥21 days). Metagenomic next-generation sequencing (mNGS) was conducted when ≥4 samples with cycle threshold values of Genome changes were consistent with the expected molecular clock of HCoV. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

Structural and Functional Analyses of the Severe Acute Respiratory Syndrome Coronavirus Endoribonuclease Nsp15

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Bhardwaj, Kanchan; Palaninathan, Satheesh; Alcantara, Joanna Maria Ortiz; Yi, Lillian Li; Guarino, Linda; Sacchettini, James C.; Kao, C. Cheng (TAM)

2008-03-31

The severe acute respiratory syndrome (SARS) coronavirus encodes several RNA-processing enzymes that are unusual for RNA viruses, including Nsp15 (nonstructural protein 15), a hexameric endoribonuclease that preferentially cleaves 3' of uridines. We solved the structure of a catalytically inactive mutant version of Nsp15, which was crystallized as a hexamer. The structure contains unreported flexibility in the active site of each subunit. Substitutions in the active site residues serine 293 and proline 343 allowed Nsp15 to cleave at cytidylate, whereas mutation of leucine 345 rendered Nsp15 able to cleave at purines as well as pyrimidines. Mutations that targeted the residues involved in subunit interactions generally resulted in the formation of catalytically inactive monomers. The RNA-binding residues were mapped by a method linking reversible cross-linking, RNA affinity purification, and peptide fingerprinting. Alanine substitution of several residues in the RNA-contacting portion of Nsp15 did not affect hexamer formation but decreased the affinity of RNA binding and reduced endonuclease activity. This suggests a model for Nsp15 hexamer interaction with RNA.

Knowledge and attitude towards the Middle East respiratory syndrome coronavirus among healthcare personnel in the southern region of Saudi Arabia.

Science.gov (United States)

Abbag, Huda F; El-Mekki, Awad Ahmed; Al Bshabshe, Ali Aobaid Ali; Mahfouz, Ahmed A; Al-Dosry, Ahasen A; Mirdad, Rasha T; AlKhttabi, Nora F; Abbag, Lubna F

2018-03-07

Middle East respiratory syndrome coronavirus (MERS-CoV) belongs to the family Coronaviridae, and is named for the crown-like spikes on its surface. The clinical presentation of MERS-CoV infection ranges from asymptomatic to very severe disease, and the classical presentation includes fever, cough chills, sore throat, myalgia, and arthralgia. A cross-sectional study of 339 healthcare personnel was conducted over an 8-month period in the Aseer region of Saudi Arabia using a structured survey that included demographic information and questions testing participant's knowledge. Approximately two-thirds of the respondents properly identified the causative agent of MERS-CoV as an RNA virus (66.4%, n=225) that is enveloped (68.1%, n=231). On the other hand, few respondents identified the proper number of strains or the genus (16.5% and 17.4%, respectively). More than half of the study sample identified the disease as zoonotic (57.2%, n=194). Similarly, 89.1% (n=302) identified that camels and bats are prone to infection with coronaviruses. Only 23.9% (n=81) properly identified March through May as the season with the highest transmission rate. There was a massive lack of adequate knowledge regarding prevalence of antibodies. Only 18.3% (n=62) of respondents identified PCR as the proper diagnostic confirmatory test for MERS-CoV infection. Regarding MERS-CoV clinical features, 76.4% (n=259) recognized the presence of sub-clinical infection, 64.7% (n=218) indicated that cases should be immediately isolated, and 46.9% (n=159) identified the main cause of mortality as respiratory failure. There is limited microbiological and virological knowledge of MERS-CoV infection among healthcare personnel in the southern region of Saudi Arabia, although the clinical aspects are known. Copyright © 2018. Published by Elsevier Ltd.

An Opportunistic Pathogen Afforded Ample Opportunities: Middle East Respiratory Syndrome Coronavirus

Directory of Open Access Journals (Sweden)

Ian M. Mackay

2017-12-01

Full Text Available The human coronaviruses (CoV include HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, some of which have been known for decades. The severe acute respiratory syndrome (SARS CoV briefly emerged into the human population but was controlled. In 2012, another novel severely human pathogenic CoV—the Middle East Respiratory Syndrome (MERS-CoV—was identified in the Kingdom of Saudi Arabia; 80% of over 2000 human cases have been recorded over five years. Targeted research remains key to developing control strategies for MERS-CoV, a cause of mild illness in its camel reservoir. A new therapeutic toolbox being developed in response to MERS is also teaching us more about how CoVs cause disease. Travel-related cases continue to challenge the world’s surveillance and response capabilities, and more data are needed to understand unexplained primary transmission. Signs of genetic change have been recorded, but it remains unclear whether there is any impact on clinical disease. How camels came to carry the virus remains academic to the control of MERS. To date, human-to-human transmission has been inefficient, but virus surveillance, characterisation, and reporting are key to responding to any future change. MERS-CoV is not currently a pandemic threat; it is spread mainly with the aid of human habit and error.

Non-kinetic capabilities: complementing the kinetic prevalence to targeting

OpenAIRE

Ducheine, P.

2014-01-01

Targeting is used in military doctrine to describe a military operational way, using (military) means to influence a target (or addressee) in order to achieve designated political and/or military goals. The four factors italicized are used to analyse non-kinetic targeting, complementing our knowledge and understanding of the kinetic prevalence. Paradoxically, non-kinetic targeting is not recognized as a separate concept: kinetic and non-kinetic are intertwined facets of targeting. Kinetic tar...

Absence of E protein arrests transmissible gastroenteritis coronavirus maturation in the secretory pathway

International Nuclear Information System (INIS)

Ortego, Javier; Ceriani, Juan E.; Patino, Cristina; Plana, Juan; Enjuanes, Luis

2007-01-01

A recombinant transmissible gastroenteritis coronavirus (rTGEV) in which E gene was deleted (rTGEV-ΔE) has been engineered. This deletion mutant only grows in cells expressing E protein (E + cells) indicating that E was an essential gene for TGEV replication. Electron microscopy studies of rTGEV-ΔE infected BHK-pAPN-E - cells showed that only immature intracellular virions were assembled. These virions were non-infectious and not secreted to the extracellular medium in BHK-pAPN-E - cells. RNA and protein composition analysis by RNase-gold and immunoelectron microscopy showed that rTGEV-ΔE virions contained RNA and also all the structural TGEV proteins, except the deleted E protein. Nevertheless, full virion maturation was blocked. Studies of the rTGEV-ΔE subcellular localization by confocal and immunoelectron microscopy in infected E - cells showed that in the absence of E protein virus trafficking was arrested in the intermediate compartment. Therefore, the absence of E protein in TGEV resulted in two actions, a blockade of virus trafficking in the membranes of the secretory pathway, and prevention of full virus maturation

The Characteristics of Middle Eastern Respiratory Syndrome Coronavirus Transmission Dynamics in South Korea.

Science.gov (United States)

Kim, Yunhwan; Lee, Sunmi; Chu, Chaeshin; Choe, Seoyun; Hong, Saeme; Shin, Youngseo

2016-02-01

The outbreak of Middle Eastern respiratory syndrome coronavirus (MERS-CoV) was one of the major events in South Korea in 2015. In particular, this study pays attention to formulating a mathematical model for MERS transmission dynamics and estimating transmission rates. Incidence data of MERS-CoV from the government authority was analyzed for the first aim and a mathematical model was built and analyzed for the second aim of the study. A mathematical model for MERS-CoV transmission dynamics is used to estimate the transmission rates in two periods due to the implementation of intensive interventions. Using the estimates of the transmission rates, the basic reproduction number was estimated in two periods. Due to the superspreader, the basic reproduction number was very large in the first period; however, the basic reproduction number of the second period has reduced significantly after intensive interventions. It turned out to be the intensive isolation and quarantine interventions that were the most critical factors that prevented the spread of the MERS outbreak. The results are expected to be useful to devise more efficient intervention strategies in the future.

Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection

Science.gov (United States)

Millet, Jean Kaoru; Whittaker, Gary R.

2016-01-01

Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay. PMID:28018942

Prevalence of comorbidities in the Middle East respiratory syndrome coronavirus (MERS-CoV): a systematic review and meta-analysis.

Science.gov (United States)

Badawi, Alaa; Ryoo, Seung Gwan

2016-08-01

The Middle East respiratory syndrome coronavirus (MERS-CoV) is associated with life-threatening severe illnesses and a mortality rate of approximately 35%, particularly in patients with underlying comorbidities. A systematic analysis of 637 MERS-CoV cases suggests that diabetes and hypertension are equally prevalent in approximately 50% of the patients. Cardiac diseases are present in 30% and obesity in 16% of the cases. These conditions down-regulate the synthesis of proinflammatory cytokines and impair the host's innate and humoral immune systems. In conclusion, protection against MERS-CoV and other respiratory infections can be improved if public health vaccination strategies are tailored to target persons with chronic disorders. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Origin and Possible Genetic Recombination of the Middle East Respiratory Syndrome Coronavirus from the First Imported Case in China: Phylogenetics and Coalescence Analysis.

Science.gov (United States)

Wang, Yanqun; Liu, Di; Shi, Weifeng; Lu, Roujian; Wang, Wenling; Zhao, Yanjie; Deng, Yao; Zhou, Weimin; Ren, Hongguang; Wu, Jun; Wang, Yu; Wu, Guizhen; Gao, George F; Tan, Wenjie

2015-09-08

The Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe acute respiratory tract infection with a high fatality rate in humans. Coronaviruses are capable of infecting multiple species and can evolve rapidly through recombination events. Here, we report the complete genomic sequence analysis of a MERS-CoV strain imported to China from South Korea. The imported virus, provisionally named ChinaGD01, belongs to group 3 in clade B in the whole-genome phylogenetic tree and also has a similar tree topology structure in the open reading frame 1a and -b (ORF1ab) gene segment but clusters with group 5 of clade B in the tree constructed using the S gene. Genetic recombination analysis and lineage-specific single-nucleotide polymorphism (SNP) comparison suggest that the imported virus is a recombinant comprising group 3 and group 5 elements. The time-resolved phylogenetic estimation indicates that the recombination event likely occurred in the second half of 2014. Genetic recombination events between group 3 and group 5 of clade B may have implications for the transmissibility of the virus. The recent outbreak of MERS-CoV in South Korea has attracted global media attention due to the speed of spread and onward transmission. Here, we present the complete genome of the first imported MERS-CoV case in China and demonstrate genetic recombination events between group 3 and group 5 of clade B that may have implications for the transmissibility of MERS-CoV. Copyright © 2015 Wang et al.

Expression analysis of miRNA and target mRNAs in esophageal cancer

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Meng, X.R. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China); Lu, P. [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Mei, J.Z.; Liu, G.J. [Medical Oncology Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Fan, Q.X. [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China)

2014-08-01

We aimed to investigate miRNAs and related mRNAs through a network-based approach in order to learn the crucial role that they play in the biological processes of esophageal cancer. Esophageal squamous-cell carcinoma (ESCC) and adenocarcinoma (EAC)-related miRNA and gene expression data were downloaded from the Gene Expression Omnibus database, and differentially expressed miRNAs and genes were selected. Target genes of differentially expressed miRNAs were predicted and their regulatory networks were constructed. Differentially expressed miRNA analysis selected four miRNAs associated with EAC and ESCC, among which hsa-miR-21 and hsa-miR-202 were shared by both diseases. hsa-miR-202 was reported for the first time to be associated with esophageal cancer in the present study. Differentially expressed miRNA target genes were mainly involved in cancer-related and signal-transduction pathways. Functional categories of these target genes were related to transcriptional regulation. The results may indicate potential target miRNAs and genes for future investigations of esophageal cancer.

Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia.

Science.gov (United States)

Sabir, Jamal S M; Lam, Tommy T-Y; Ahmed, Mohamed M M; Li, Lifeng; Shen, Yongyi; Abo-Aba, Salah E M; Qureshi, Muhammd I; Abu-Zeid, Mohamed; Zhang, Yu; Khiyami, Mohammad A; Alharbi, Njud S; Hajrah, Nahid H; Sabir, Meshaal J; Mutwakil, Mohammed H Z; Kabli, Saleh A; Alsulaimany, Faten A S; Obaid, Abdullah Y; Zhou, Boping; Smith, David K; Holmes, Edward C; Zhu, Huachen; Guan, Yi

2016-01-01

Outbreaks of Middle East respiratory syndrome (MERS) raise questions about the prevalence and evolution of the MERS coronavirus (CoV) in its animal reservoir. Our surveillance in Saudi Arabia in 2014 and 2015 showed that viruses of the MERS-CoV species and a human CoV 229E-related lineage co-circulated at high prevalence, with frequent co-infections in the upper respiratory tract of dromedary camels. viruses of the betacoronavirus 1 species, we found that dromedary camels share three CoV species with humans. Several MERS-CoV lineages were present in camels, including a recombinant lineage that has been dominant since December 2014 and that subsequently led to the human outbreaks in 2015. Camels therefore serve as an important reservoir for the maintenance and diversification of the MERS-CoVs and are the source of human infections with this virus. Copyright © 2016, American Association for the Advancement of Science.

Overview of preparedness and response for Middle East respiratory syndrome coronavirus (MERS-CoV) in Oman.

Science.gov (United States)

Al-Abaidani, I S; Al-Maani, A S; Al-Kindi, H S; Al-Jardani, A K; Abdel-Hady, D M; Zayed, B E; Al-Harthy, K S; Al-Shaqsi, K H; Al-Abri, S S

2014-12-01

Several countries in the Middle East and around 22 countries worldwide have reported cases of human infection with the Middle East respiratory syndrome coronavirus (MERS-CoV). The exceptionally high fatality rate resulting from MERS-CoV infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. Within the framework of the national acute respiratory illness surveillance, the Ministry of Health in the Sultanate of Oman has announced two confirmed cases of MERS-CoV to date. The aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. The absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in Oman. Copyright © 2014. Published by Elsevier Ltd.

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

International Nuclear Information System (INIS)

Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian

2007-01-01

The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection

Overview of preparedness and response for Middle East respiratory syndrome coronavirus (MERS-CoV in Oman

Directory of Open Access Journals (Sweden)

I.S. Al-Abaidani

2014-12-01

Full Text Available Several countries in the Middle East and around 22 countries worldwide have reported cases of human infection with the Middle East respiratory syndrome coronavirus (MERS-CoV. The exceptionally high fatality rate resulting from MERS-CoV infection in conjunction with the paucity of knowledge about this emerging virus has led to major public and international concern. Within the framework of the national acute respiratory illness surveillance, the Ministry of Health in the Sultanate of Oman has announced two confirmed cases of MERS-CoV to date. The aim of this report is to describe the epidemiological aspects of these two cases and to highlight the importance of public health preparedness and response. The absence of secondary cases among contacts of the reported cases can be seen as evidence of the effectiveness of infection prevention and control precautions as an important pillar of the national preparedness and response plan applied in the health care institutions in Oman.

Human coronavirus and severe acute respiratory infection in Southern Brazil.

Science.gov (United States)

Trombetta, Hygor; Faggion, Heloisa Z; Leotte, Jaqueline; Nogueira, Meri B; Vidal, Luine R R; Raboni, Sonia M

2016-05-01

Human coronaviruses (HCoVs) are an important cause of respiratory tract infection and are responsible for causing the common cold in the general population. Thus, adequate surveillance of HCoV is essential. This study aimed to analyze the impact of HCoV infections and their relation to severe acute respiratory infection (SARI) in a hospitalized population in Southern Brazil. A cross-sectional study was conducted at a tertiary care hospital, and assessed inpatients under investigation for SARI by the hospital epidemiology department, and all patients who had nasopharyngeal aspirates collected from January 2012 to December 2013 to detect respiratory viruses (RVs). Viral infection was detected by multiplex reverse transcriptase polymerase chain reaction (RT-PCR), with primers specific to the subtypes HCoV-229E/NL63 and OC43/HKU1. The overall positivity rate was 58.8% (444/755), and HCoVs were detected in 7.6% (n = 34) of positive samples. Children below two years of age were most frequently affected (62%). Comorbidities were more likely to be associated with HCoVs than with other RVs. Immunosuppression was an independent risk factor for HCoV infection (OR = 3.5, 95% CI 1.6-7.6). Dyspnea was less frequently associated with HCoV infection (p infected with HCoV (9%) died from respiratory infection. HCoVs are important respiratory pathogens, especially in hospitalized children under 2 years of age and in immunosuppressed patients. They may account for a small proportion of SARI diagnoses, increased need for mechanical ventilation, intensive care unit admission, and death.

Human thyroid peroxidase: complete cDNA and protein sequence, chromosome mapping, and identification of two alternately spliced mRNAs

International Nuclear Information System (INIS)

Kimura, S.; Kotani, T.; McBride, O.W.; Umeki, K.; Hirai, K.; Nakayama, T.; Ohtaki, S.

1987-01-01

Two forms of human thyroid peroxidase cDNAs were isolated from a λgt11 cDNA library, prepared from Graves disease thyroid tissue mRNA, by use of oligonucleotides. The longest complete cDNA, designated phTPO-1, has 3048 nucleotides and an open reading frame consisting of 933 amino acids, which would encode a protein with a molecular weight of 103,026. Five potential asparagine-linked glycosylation sites are found in the deduced amino acid sequence. The second peroxidase cDNA, designated phTPO-2, is almost identical to phTPO-1 beginning 605 base pairs downstream except that it contains 1-base-pair difference and lacks 171 base pairs in the middle of the sequence. This results in a loss of 57 amino acids corresponding to a molecular weight of 6282. Interestingly, this 171-nucleotide sequence has GT and AG at its 5' and 3' boundaries, respectively, that are in good agreement with donor and acceptor splice site consensus sequences. Using specific oligonucleotide probes for the mRNAs derived from the cDNA sequences hTOP-1 and hTOP-2, the authors show that both are expressed in all thyroid tissues examined and the relative level of two mRNAs is different in each sample. The results suggest that two thyroid peroxidase proteins might be generated through alternate splicing of the same gene. By using somatic cell hybrid lines, the thyroid peroxidase gene was mapped to the short arm of human chromosome 2

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

International Nuclear Information System (INIS)

Haan, Cornelis A.M. de; Wit, Marel de; Kuo, Lili; Montalto-Morrison, Cynthia; Haagmans, Bart L.; Weiss, Susan R.; Masters, Paul S.; Rottier, Peter J.M.

2003-01-01

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon

Peptide Mimicrying Between SARS Coronavirus Spike Protein and Human Proteins Reacts with SARS Patient Serum

Directory of Open Access Journals (Sweden)

K.-Y. Hwa

2008-01-01

Full Text Available Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells. Severe acute respiratory syndrome (SARS is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV. The spike (S protein of SARS-CoV plays an important role in the virus entry into a cell. In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins. Two of the peptides D07 (residues 927–937 and D08 (residues 942–951 were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490–502 stimulated A549 to proliferate and secrete IL-8. The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.

Immune responses against SARS-coronavirus nucleocapsid protein induced by DNA vaccine

International Nuclear Information System (INIS)

Zhao Ping; Cao Jie; Zhao Lanjuan; Qin Zhaolin; Ke Jinshan; Pan Wei; Ren Hao; Yu Jianguo; Qi Zhongtian

2005-01-01

The nucleocapsid (N) protein of SARS-coronavirus (SARS-CoV) is the key protein for the formation of the helical nucleocapsid during virion assembly. This protein is believed to be more conserved than other proteins of the virus, such as spike and membrane glycoprotein. In this study, the N protein of SARS-CoV was expressed in Escherichia coli DH5α and identified with pooled sera from patients in the convalescence phase of SARS. A plasmid pCI-N, encoding the full-length N gene of SARS-CoV, was constructed. Expression of the N protein was observed in COS1 cells following transfection with pCI-N. The immune responses induced by intramuscular immunization with pCI-N were evaluated in a murine model. Serum anti-N immunoglobulins and splenocytes proliferative responses against N protein were observed in immunized BALB/c mice. The major immunoglobulin G subclass recognizing N protein was immunoglobulin G2a, and stimulated splenocytes secreted high levels of gamma interferon and IL-2 in response to N protein. More importantly, the immunized mice produced strong delayed-type hypersensitivity (DTH) and CD8 + CTL responses to N protein. The study shows that N protein of SARS-CoV not only is an important B cell immunogen, but also can elicit broad-based cellular immune responses. The results indicate that the N protein may be of potential value in vaccine development for specific prophylaxis and treatment against SARS

Evaluation of a multiplex immunoassay for bovine respiratory syncytial virus and bovine coronavirus antibodies in bulk tank milk against two indirect ELISAs using latent class analysis

DEFF Research Database (Denmark)

Toftaker, Ingrid; Toft, Nils; Stokstad, Maria

2018-01-01

Bovine respiratory syncytial virus (BRSV) and bovine coronavirus (BCV) are responsible for respiratory disease and diarrhea in cattle worldwide. The Norwegian control program against these infections is based on herd-level diagnosis using a new multiplex immunoassay. The objective of this study...... was to estimate sensitivity and specificity across different cut-off values for the MVD-Enferplex BCV/BRSV multiplex, by comparing them to a commercially available ELISA, the SVANOVIR® BCV-Ab and SVANOVIR® BRSV-Ab, respectively. We analyzed bulk tank milk samples from 360 herds in a low- and 360 herds in a high...

Roadmap to developing a recombinant coronavirus S protein receptor-binding domain vaccine for severe acute respiratory syndrome

Science.gov (United States)

Jiang, Shibo; Bottazzi, Maria Elena; Du, Lanying; Lustigman, Sara; Tseng, Chien-Te Kent; Curti, Elena; Jones, Kathryn; Zhan, Bin; Hotez, Peter J

2013-01-01

A subunit vaccine, RBD-S, is under development to prevent severe acute respiratory syndrome (SARS) caused by SARS coronavirus (SARS-CoV), which is classified by the US NIH as a category C pathogen. This vaccine is comprised of a recombinant receptor-binding domain (RBD) of the SARS-CoV spike (S) protein and formulated on alum, together with a synthetic glucopyranosyl lipid A. The vaccine would induce neutralizing antibodies without causing Th2-type immunopathology. Vaccine development is being led by the nonprofit product development partnership; Sabin Vaccine Institute and Texas Children’s Hospital Center for Vaccine Development in collaboration with two academic partners (the New York Blood Center and University of Texas Medical Branch); an industrial partner (Immune Design Corporation); and Walter Reed Army Institute of Research. A roadmap for the product development of the RBD-S SARS vaccine is outlined with a goal to manufacture the vaccine for clinical testing within the next 5 years. PMID:23252385

Characterization of human coronavirus etiology in Chinese adults with acute upper respiratory tract infection by real-time RT-PCR assays.

Directory of Open Access Journals (Sweden)

Roujian Lu

Full Text Available BACKGROUND: In addition to SARS associated coronaviruses, 4 non-SARS related human coronaviruses (HCoVs are recognized as common respiratory pathogens. The etiology and clinical impact of HCoVs in Chinese adults with acute upper respiratory tract infection (URTI needs to be characterized systematically by molecular detection with excellent sensitivity. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we detected 4 non-SARS related HCoV species by real-time RT-PCR in 981 nasopharyngeal swabs collected from March 2009 to February 2011. All specimens were also tested for the presence of other common respiratory viruses and newly identified viruses, human metapneumovirus (hMPV and human bocavirus (HBoV. 157 of the 981 (16.0% nasopharyngeal swabs were positive for HCoVs. The species detected were 229E (96 cases, 9.8%, OC43 (42 cases, 4.3%, HKU1 (16 cases, 1.6% and NL63 (11 cases, 1.1%. HCoV-229E was circulated in 21 of the 24 months of surveillance. The detection rates for both OC43 and NL63 were showed significantly year-to-year variation between 2009/10 and 2010/11, respectively (P<0.001 and P = 0.003, and there was a higher detection frequency of HKU1 in patients aged over 60 years (P = 0.03. 48 of 157(30.57% HCoV positive patients were co-infected. Undifferentiated human rhinoviruses and influenza (Flu A were the most common viruses detected (more than 35% in HCoV co-infections. Respiratory syncytial virus (RSV, human parainfluenza virus (PIV and HBoV were detected in very low rate (less than 1% among adult patients with URTI. CONCLUSIONS/SIGNIFICANCE: All 4 non-SARS-associated HCoVs were more frequently detected by real-time RT-PCR assay in adults with URTI in Beijing and HCoV-229E led to the most prevalent infection. Our study also suggested that all non-SARS-associated HCoVs contribute significantly to URTI in adult patients in China.

Kinetic Typography

DEFF Research Database (Denmark)

van Leeuwen, Theo; Djonov, Emilia

2014-01-01

After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images.......After discussing broad cultural drivers behind the development of kinetic typography, the chapter outlines an approach to analysing kinetic typography which is based on Halliday's theory of transitivity, as applied by Kress and Van Leeuwen to visual images....

Rapid detection of MERS coronavirus-like viruses in bats: pote1ntial for tracking MERS coronavirus transmission and animal origin.

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Woo, Patrick C Y; Lau, Susanna K P; Chen, Yixin; Wong, Emily Y M; Chan, Kwok-Hung; Chen, Honglin; Zhang, Libiao; Xia, Ningshao; Yuen, Kwok-Yung

2018-03-07

Recently, we developed a monoclonal antibody-based rapid nucleocapsid protein detection assay for diagnosis of MERS coronavirus (MERS-CoV) in humans and dromedary camels. In this study, we examined the usefulness of this assay to detect other lineage C betacoronaviruses closely related to MERS-CoV in bats. The rapid MERS-CoV nucleocapsid protein detection assay was tested positive in 24 (88.9%) of 27 Tylonycteris bat CoV HKU4 (Ty-BatCoV-HKU4) RNA-positive alimentary samples of Tylonycteris pachypus and 4 (19.0%) of 21 Pipistrellus bat CoV HKU5 (Pi-BatCoV-HKU5) RNA-positive alimentary samples of Pipistrellus abramus. There was significantly more Ty-BatCoV-HKU4 RNA-positive alimentary samples than Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive by the rapid MERS-CoV nucleocapsid protein detection assay (P < 0.001 by Chi-square test). The rapid assay was tested negative in all 51 alimentary samples RNA-positive for alphacoronaviruses (Rhinolophus bat CoV HKU2, Myotis bat CoV HKU6, Miniopterus bat CoV HKU8 and Hipposideros batCoV HKU10) and 32 alimentary samples positive for lineage B (SARS-related Rhinolophus bat CoV HKU3) and lineage D (Rousettus bat CoV HKU9) betacoronaviruses. No significant difference was observed between the viral loads of Ty-BatCoV-HKU4/Pi-BatCoV-HKU5 RNA-positive alimentary samples that were tested positive and negative by the rapid test (Mann-Witney U test). The rapid MERS-CoV nucleocapsid protein detection assay is able to rapidly detect lineage C betacoronaviruses in bats. It detected significantly more Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5 because MERS-CoV is more closely related to Ty-BatCoV-HKU4 than Pi-BatCoV-HKU5. This assay will facilitate rapid on-site mass screening of animal samples for ancestors of MERS-CoV and tracking transmission in the related bat species.

Comparative Evaluation of Three Homogenization Methods for Isolating Middle East Respiratory Syndrome Coronavirus Nucleic Acids From Sputum Samples for Real-Time Reverse Transcription PCR.

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Sung, Heungsup; Yong, Dongeun; Ki, Chang Seok; Kim, Jae Seok; Seong, Moon Woo; Lee, Hyukmin; Kim, Mi Na

2016-09-01

Real-time reverse transcription PCR (rRT-PCR) of sputum samples is commonly used to diagnose Middle East respiratory syndrome coronavirus (MERS-CoV) infection. Owing to the difficulty of extracting RNA from sputum containing mucus, sputum homogenization is desirable prior to nucleic acid isolation. We determined optimal homogenization methods for isolating viral nucleic acids from sputum. We evaluated the following three sputum-homogenization methods: proteinase K and DNase I (PK-DNase) treatment, phosphate-buffered saline (PBS) treatment, and N-acetyl-L-cysteine and sodium citrate (NALC) treatment. Sputum samples were spiked with inactivated MERS-CoV culture isolates. RNA was extracted from pretreated, spiked samples using the easyMAG system (bioMérieux, France). Extracted RNAs were then subjected to rRT-PCR for MERS-CoV diagnosis (DiaPlex Q MERS-coronavirus, SolGent, Korea). While analyzing 15 spiked sputum samples prepared in technical duplicate, false-negative results were obtained with five (16.7%) and four samples (13.3%), respectively, by using the PBS and NALC methods. The range of threshold cycle (Ct) values observed when detecting upE in sputum samples was 31.1-35.4 with the PK-DNase method, 34.7-39.0 with the PBS method, and 33.9-38.6 with the NALC method. Compared with the control, which were prepared by adding a one-tenth volume of 1:1,000 diluted viral culture to PBS solution, the ranges of Ct values obtained by the PBS and NALC methods differed significantly from the mean control Ct of 33.2 (both Phomogenizing sputum samples prior to RNA extraction.

Evidence supporting a zoonotic origin of human coronavirus strain NL63.

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Huynh, Jeremy; Li, Shimena; Yount, Boyd; Smith, Alexander; Sturges, Leslie; Olsen, John C; Nagel, Juliet; Johnson, Joshua B; Agnihothram, Sudhakar; Gates, J Edward; Frieman, Matthew B; Baric, Ralph S; Donaldson, Eric F

2012-12-01

The relationship between bats and coronaviruses (CoVs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably CoVs, in the feces; however, no bat CoVs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat CoVs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (α-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCoVs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-CoVs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human CoVs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some CoVs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans.

Nucleocapsid gene analysis from an imported case of Middle East respiratory syndrome coronavirus, Malaysia

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Nor-Aziyah Mat-Rahim

2015-07-01

Full Text Available Objective: To describe the complete nucleocapsid (N gene region of Middle East respiratory syndrome coronavirus (MERS-CoV from imported case in Malaysia and the relations with human- and camel-derived MERS-CoV. Methods: Combination of throat and nasal swab specimens was subjected to viral RNA extraction. For screening, the extracted RNA was subjected to real-time RT-PCR targeting upstream of E gene, open reading frame 1b and open reading frame 1a. For confirmation, the RNA was subjected to RT-PCR targeting partial part of the RNA-dependent RNA polymerase and nucleocapsid, followed by amplification of complete N gene region. Nucleotide sequencing of the first Malaysian case of MERS-CoV was performed following the confirmation with real-time RT-PCR detection. Results: Initial analysis of partial RNA-dependent RNA polymerase and N gene revealed that the nucleotides had high similarity to Jeddah_1_2013 strain. Analysis of complete N gene region (1 242 nucleotides from the case showed high similarity and yet distinct to the nucleotide sequences of camel-derived MERS-CoV. Conclusions: From the finding, there are possibilities that the patient acquired the infection from zoonotic transmission from dromedary camels.

Exosomes released in vitro from Epstein-Barr virus (EBV)-infected cells contain EBV-encoded latent phase mRNAs.

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Canitano, Andrea; Venturi, Giulietta; Borghi, Martina; Ammendolia, Maria Grazia; Fais, Stefano

2013-09-01

EBV is a human herpesvirus associated with a number of malignancies. Both lymphoblastoid cell lines (LCLs), and EBV-infected nasopharyngeal carcinoma (NPC) cells have been demonstrated to release exosomes containing the EBV-encoded latent membrane protein 1 (LMP1), and mature micro-RNAs (EBV-miRNAs). Here we analyze the EBV protein and nucleic acid content of exosomes from different EBV-infected cells (LCL, 721 and Daudi) and we show for the first time that exosomes released from LCLs and 721 also contain EBV-encoded latent phase mRNAs. This confirms and strengthens exosomes pathogenetic potential, and might provide insights for development of novel diagnostic and therapeutic strategies. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Cesium removal and kinetics equilibrium: Precipitation kinetics

International Nuclear Information System (INIS)

Barnes, M.J.

1999-01-01

This task consisted of both non-radioactive and radioactive (tracer) tests examining the influence of potentially significant variables on cesium tetraphenylborate precipitation kinetics. The work investigated the time required to reach cesium decontamination and the conditions that affect the cesium precipitation kinetics

Middle East Respiratory Syndrome Coronavirus Nonstructural Protein 16 Is Necessary for Interferon Resistance and Viral Pathogenesis

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Menachery, Vineet D.; Gralinski, Lisa E.; Mitchell, Hugh D.; Dinnon, Kenneth H.; Leist, Sarah R.; Yount, Boyd L.; Graham, Rachel L.; McAnarney, Eileen T.; Stratton, Kelly G.; Cockrell, Adam S.; Debbink, Kari; Sims, Amy C.; Waters, Katrina M.; Baric, Ralph S.; Fernandez-Sesma, Ana

2017-11-15

Coronaviruses (CoVs) encode a mixture of highly conserved and novel genes, as well as genetic elements necessary for infection and pathogenesis, raising the possibility of common targets for attenuation and therapeutic design. In this study, we focused on highly conserved nonstructural protein 16 (NSP16), a viral 2'O-methyltransferase (2'O-MTase) that encodes critical functions in immune modulation and infection. Using reverse genetics, we disrupted a key motif in the conserved KDKE motif of Middle East respiratory syndrome CoV (MERS-CoV) NSP16 (D130A) and evaluated the effect on viral infection and pathogenesis. While the absence of 2'O-MTase activity had only a marginal impact on propagation and replication in Vero cells, dNSP16 mutant MERS-CoV demonstrated significant attenuation relative to the control both in primary human airway cell cultures andin vivo. Further examination indicated that dNSP16 mutant MERS-CoV had a type I interferon (IFN)-based attenuation and was partially restored in the absence of molecules of IFN-induced proteins with tetratricopeptide repeats. Importantly, the robust attenuation permitted the use of dNSP16 mutant MERS-CoV as a live attenuated vaccine platform protecting from a challenge with a mouse-adapted MERS-CoV strain. These studies demonstrate the importance of the conserved 2'O-MTase activity for CoV pathogenesis and highlight NSP16 as a conserved universal target for rapid live attenuated vaccine design in an expanding CoV outbreak setting.

IMPORTANCECoronavirus (CoV) emergence in both humans and livestock represents a significant threat to global public health, as evidenced by the sudden emergence of severe acute respiratory syndrome CoV (SARS-CoV), MERS-CoV, porcine epidemic diarrhea virus, and swine delta CoV in the 21st century. These studies describe an approach that

Taking forward a ‘One Health’ approach for turning the tide against the Middle East respiratory syndrome coronavirus and other zoonotic pathogens with epidemic potential

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Alimuddin Zumla

2016-06-01

Full Text Available The appearance of novel pathogens of humans with epidemic potential and high mortality rates have threatened global health security for centuries. Over the past few decades new zoonotic infectious diseases of humans caused by pathogens arising from animal reservoirs have included West Nile virus, Yellow fever virus, Ebola virus, Nipah virus, Lassa Fever virus, Hanta virus, Dengue fever virus, Rift Valley fever virus, Crimean-Congo haemorrhagic fever virus, severe acute respiratory syndrome coronavirus, highly pathogenic avian influenza viruses, Middle East Respiratory Syndrome Coronavirus, and Zika virus. The recent Ebola Virus Disease epidemic in West Africa and the ongoing Zika Virus outbreak in South America highlight the urgent need for local, regional and international public health systems to be be more coordinated and better prepared. The One Health concept focuses on the relationship and interconnectedness between Humans, Animals and the Environment, and recognizes that the health and wellbeing of humans is intimately connected to the health of animals and their environment (and vice versa. Critical to the establishment of a One Health platform is the creation of a multidisciplinary team with a range of expertise including public health officers, physicians, veterinarians, animal husbandry specialists, agriculturalists, ecologists, vector biologists, viral phylogeneticists, and researchers to co-operate, collaborate to learn more about zoonotic spread between animals, humans and the environment and to monitor, respond to and prevent major outbreaks. We discuss the unique opportunities for Middle Eastern and African stakeholders to take leadership in building equitable and effective partnerships with all stakeholders involved in human and health systems to take forward a ‘One Health’ approach to control such zoonotic pathogens with epidemic potential.

Treatment outcomes for patients with Middle Eastern Respiratory Syndrome Coronavirus (MERS CoV) infection at a coronavirus referral center in the Kingdom of Saudi Arabia.

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Al Ghamdi, Mohammed; Alghamdi, Khalid M; Ghandoora, Yasmeen; Alzahrani, Ameera; Salah, Fatmah; Alsulami, Abdulmoatani; Bawayan, Mayada F; Vaidya, Dhananjay; Perl, Trish M; Sood, Geeta

2016-04-21

Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV) is a poorly understood disease with no known treatments. We describe the clinical features and treatment outcomes of patients with laboratory confirmed MERS-CoV at a regional referral center in the Kingdom of Saudi Arabia. In 2014, a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of MERS-CoV to determine clinical and treatment characteristics associated with death. Confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. Fifty-one patients had an overall mortality of 37 %. Most patients were male (78 %) with a mean age of 54 years. Almost a quarter of the patients were healthcare workers (23.5 %) and 41 % had a known exposure to another person with MERS-CoV. Survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (ALT), clearance of MERS-CoV on repeat PCR polymerase chain reaction (PCR) testing, and mycophenolate mofetil treatment. Survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, CXR (chest X-ray) abnormalities, leukocytosis, creatinine >1 · 5 mg/dL, thrombocytopenia, anemia, and renal failure. In a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. Care for patients with MERS-CoV remains a challenge. In this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. Severity of illness was the greatest predictor of reduced survival in the multivariate analysis. Larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for MERS-CoV.

Expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 in cocultures of dissociated DRG neurons and skeletal muscle cells in administration of NGF or NT-3

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Weiwei Zhang

2012-07-01

Full Text Available Both neurotrophins (NTs and target skeletal muscle (SKM cells are essential for the maintenance of the function of neurons and nerve-muscle communication. However, much less is known about the association of target SKM cells with distinct NTs on the expression of mRNAs for preprotachykinin (PPT, calcitonin-gene related peptide (CGRP, neurofilament 200 (NF-200, and microtubule associated protein 2 (MAP-2 in dorsal root ganglion (DRG sensory neurons. In the present study, a neuromuscular coculture model of dissociated dorsal root ganglion (DRG neurons and SKM cells was established. The morphology of DRG neurons and SKM cells in coculture was observed with an inverted phase contrast microscope. The effects of nerve growth factor (NGF or neurotrophin-3 (NT-3 on the expression of mRNAs for PPT, CGRP, NF-200, and MAP-2 was analyzed by real time-PCR assay. The morphology of DRG neuronal cell bodies and SKM cells in neuromuscular coculture at different conditions was similar. The neurons presented evidence of dense neurite outgrowth in the presence of distinct NTs in neuromuscular cocultures. NGF and NT-3 increased mRNA levels of PPT, CGRP, and NF-200, but not MAP-2, in neuromuscular cocultures. These results offer new clues towards a better understanding of the association of target SKM cells with distinct NTs on the expression of mRNAs for PPT, CGRP, NF-200 and MAP-2, and implicate the association of target SKM cells and NTs with DRG sensory neuronal phenotypes.

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

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Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Exosomes enriched in stemness/metastatic-related mRNAS promote oncogenic potential in breast cancer.

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Rodríguez, Marta; Silva, Javier; Herrera, Alberto; Herrera, Mercedes; Peña, Cristina; Martín, Paloma; Gil-Calderón, Beatriz; Larriba, María Jesús; Coronado, M Josés; Soldevilla, Beatriz; Turrión, Víctor S; Provencio, Mariano; Sánchez, Antonio; Bonilla, Félix; García-Barberán, Vanesa

2015-12-01

Cancer cells efficiently transfer exosome contents (essentially mRNAs and microRNAs) to other cell types, modifying immune responses, cell growth, angiogenesis and metastasis. Here we analyzed the exosomes release by breast tumor cells with different capacities of stemness/metastasis based on CXCR4 expression, and evaluated their capacity to generate oncogenic features in recipient cells. Breast cancer cells overexpressing CXCR4 showed an increase in stemness-related markers, and in proliferation, migration and invasion capacities. Furthermore, recipient cells treated with exosomes from CXCR4-cells showed increased in the same abilities. Moreover, inoculation of CXCR4-cell-derived exosomes in immunocompromised mice stimulated primary tumor growth and metastatic potential. Comparison of nucleic acids contained into exosomes isolated from patients revealed a "stemness and metastatic" signature in exosomes of patients with worse prognosis. Finally, our data supported the view that cancer cells with stem-like properties show concomitant metastatic behavior, and their exosomes stimulate tumor progression and metastasis. Exosomes-derived nucleic acids from plasma of breast cancer patients are suitable markers in the prognosis of such patients.

Heparin kinetics

International Nuclear Information System (INIS)

Swart, C.A.M. de.

1983-01-01

The author has studied the kinetics of heparin and heparin fractions after intravenous administration in humans and in this thesis the results of this study are reported. Basic knowledge about the physico-chemical properties of heparin and its interactions with proteins resulting in anticoagulant and lipolytic effects are discussed in a review (chapter II), which also comprises some clinical aspects of heparin therapy. In chapter III the kinetics of the anticoagulant effect are described after intravenous administration of five commercial heparin preparations. A mathematical model is presented that fits best to these kinetics. The kinetics of the anticoagulant and lipolytic effects after intravenous injection of various 35 S-radiolabelled heparin fractions and their relationship with the disappearance of the radiolabel are described in chapter IV. Chapter V gives a description of the kinetics of two radiolabels after injection of in vitro formed complexes consisting of purified, 125 I-radiolabelled antithrombin III and various 35 S-radiolabelled heparin fractions. (Auth.)

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: Engineering a recombination-resistant genome

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Yount, Boyd; Roberts, Rhonda S.; Lindesmith, Lisa; Baric, Ralph S.

2006-08-01

Live virus vaccines provide significant protection against many detrimental human and animal diseases, but reversion to virulence by mutation and recombination has reduced appeal. Using severe acute respiratory syndrome coronavirus as a model, we engineered a different transcription regulatory circuit and isolated recombinant viruses. The transcription network allowed for efficient expression of the viral transcripts and proteins, and the recombinant viruses replicated to WT levels. Recombinant genomes were then constructed that contained mixtures of the WT and mutant regulatory circuits, reflecting recombinant viruses that might occur in nature. Although viable viruses could readily be isolated from WT and recombinant genomes containing homogeneous transcription circuits, chimeras that contained mixed regulatory networks were invariantly lethal, because viable chimeric viruses were not isolated. Mechanistically, mixed regulatory circuits promoted inefficient subgenomic transcription from inappropriate start sites, resulting in truncated ORFs and effectively minimize viral structural protein expression. Engineering regulatory transcription circuits of intercommunicating alleles successfully introduces genetic traps into a viral genome that are lethal in RNA recombinant progeny viruses. regulation | systems biology | vaccine design

Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

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Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

2009-01-01

The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

Increase in Middle East Respiratory Syndrome-Coronavirus Cases in Saudi Arabia Linked to Hospital Outbreak With Continued Circulation of Recombinant Virus, July 1–August 31, 2015

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Assiri, Abdullah M.; Biggs, Holly M.; Abedi, Glen R.; Lu, Xiaoyan; Bin Saeed, Abdulaziz; Abdalla, Osman; Mohammed, Mutaz; Al-Abdely, Hail M.; Algarni, Homoud S.; Alhakeem, Raafat F.; Almasri, Malak M.; Alsharef, Ali A.; Nooh, Randa; Erdman, Dean D.; Gerber, Susan I.; Watson, John T.

2016-01-01

During July–August 2015, the number of cases of Middle East respiratory syndrome (MERS) reported from Saudi Arabia increased dramatically. We reviewed the 143 confirmed cases from this period and classified each based upon likely transmission source. We found that the surge in cases resulted predominantly (90%) from secondary transmission largely attributable to an outbreak at a single healthcare facility in Riyadh. Genome sequencing of MERS coronavirus from 6 cases demonstrated continued circulation of the recently described recombinant virus. A single unique frameshift deletion in open reading frame 5 was detected in the viral sequence from 1 case. PMID:27704019

Comparison of Bovine coronavirus-specific and Bovine respiratory syncytial virus-specific antibodies in serum versus milk samples detected by enzyme-linked immunosorbent assay.

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Ohlson, Anna; Blanco-Penedo, Isabel; Fall, Nils

2014-01-01

Bovine coronavirus (BCV; Betacoronavirus 1) and Bovine respiratory syncytial virus (BRSV) are significant causes of enteric and respiratory disease in beef and dairy cattle throughout the world. Indirect enzyme-linked immunosorbent assays are widely used to detect serum antibodies for herd monitoring and prevalence studies. In dairy herds, milk is more readily collected than serum. Hence, in order to investigate the test agreement between serum and milk, both serum and milk samples from 105 cows in 27 dairy herds were analyzed in parallel for presence of immunoglobulin G antibodies to BCV and BRSV. The Bland-Altman analyses of data demonstrated good agreement between serum and milk antibody titers for both viruses. The results indicate milk samples are sufficient for surveillance of antibodies to BCV and BRSV.

Two deletion variants of Middle East respiratory syndrome coronavirus found in a patient with characteristic symptoms.

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Xie, Qian; Cao, Yujuan; Su, Juan; Wu, Jie; Wu, Xianbo; Wan, Chengsong; He, Mingliang; Ke, Changwen; Zhang, Bao; Zhao, Wei

2017-08-01

Significant sequence variation of Middle East respiratory syndrome coronavirus (MERS CoV) has never been detected since it was first reported in 2012. A MERS patient came from Korea to China in late May 2015. The patient was 44 years old and had symptoms including high fever, dry cough with a little phlegm, and shortness of breath, which are roughly consistent with those associated with MERS, and had had close contact with individuals with confirmed cases of MERS.After one month of therapy with antiviral, anti-infection, and immune-enhancing agents, the patient recovered in the hospital and was discharged. A nasopharyngeal swab sample was collected for direct sequencing, which revealed two deletion variants of MERS CoV. Deletions of 414 and 419 nt occurred between ORF5 and the E protein, resulting in a partial protein fusion or truncation of ORF5 and the E protein. Functional analysis by bioinformatics and comparison to previous studies implied that the two variants might be defective in their ability to package MERS CoV. However, the mechanism of how these deletions occurred and what effects they have need to be further investigated.

Expression of beta-keratin mRNAs and proline uptake in epidermal cells of growing scales and pad lamellae of gecko lizards

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Alibardi, Lorenzo; Toni, Mattia; Valle, Luisa Dalla

2007-01-01

Beta-keratins form a large part of the proteins contained in the hard beta layer of reptilian scales. The expression of genes encoding glycine–proline-rich beta-keratins in normal and regenerating epidermis of two species of gecko lizards has been studied by in situ hybridization. The probes localize mRNAs in differentiating oberhautchen and beta cells of growing scales and in modified scales, termed pad lamellae, on the digits of gecko lizards. In situ localization at the ultrastructural level shows clusters of gold particles in the cytoplasm among beta-keratin filaments of oberhautchen and beta cells. They are also present in the differentiating elongation or setae of oberhautchen cells present in pad lamellae. Setae allow geckos to adhere and climb vertical surfaces. Oberhautchen and beta cells also incorporate tritiated proline. The fine localization of the beta-keratin mRNAs and the uptake of proline confirms the biomolecular data that identified glycine–proline-rich beta-keratin in differentiating beta cells of gecko epidermis. The present study also shows the presence of differentiating and metabolically active cells in both inner and outer oberhautchen/beta cells at the base of the outer setae localized at the tip of pad lamellae. The addition of new beta and alpha cells to the corneous layer near the tip of the outer setae explains the anterior movement of the setae along the apical free-margin of pad lamellae. The rapid replacement of setae ensures the continuous usage of the gecko's adhesive devices, the pad lamellae, during most of their active life. PMID:17553098

Virological and serological analysis of a recent Middle East respiratory syndrome coronavirus infection case on a triple combination antiviral regimen.

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Spanakis, Nikolaos; Tsiodras, Sotirios; Haagmans, Bart L; Raj, V Stalin; Pontikis, Kostantinos; Koutsoukou, Antonia; Koulouris, Nikolaos G; Osterhaus, Albert D M E; Koopmans, Marion P G; Tsakris, Athanassios

2014-12-01

Serological, molecular and phylogenetic analyses of a recently imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) in Greece are reported. Although MERS-CoV remained detectable in the respiratory tract secretions of the patient until the fourth week of illness, viraemia was last detected 2 days after initiation of triple combination therapy with pegylated interferon, ribavirin and lopinavir/ritonavir, administered from Day 13 of illness. Phylogenetic analysis of the virus showed close similarity with other human MERS-CoVs from the recent Jeddah outbreak in Saudi Arabia. Immunoglobulin G (IgG) titres peaked 3 weeks after the onset of illness, whilst IgM levels remained constantly elevated during the follow-up period (second to fifth week of illness). Serological testing confirmed by virus neutralisation assay detected an additional case that was a close contact of the patient. Copyright © 2014. Published by Elsevier B.V.

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

International Nuclear Information System (INIS)

Collomb, J.; Finance, C.; Alabouch, S.; Laporte, J.

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabelling with 32 P and enzymatic labelling through covalent linkage to peroxidase and chemiluminescence detection. The radioactive probe 174 detected as little as 1 to 3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in faecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors)

Radioactive and enzymatic cloned cDNA probes for bovine enteric coronavirus detection by molecular hybridization

Energy Technology Data Exchange (ETDEWEB)

Collomb, J; Finance, C; Alabouch, S [Lab. de Microbiologie Moleculaire, Faculte des Sciences Pharmaceutiques et Biologiques, Univ. de Nancy I, Nancy (France); Laporte, J [Station de Virologie et d' Immunologie Moleculaires, INRA, Jouy-en-Josas (France)

1992-01-01

Genomic RNA of F15 strain bovine enteric coronavirus (BECV) was cloned in E. coli. Three clones (174, 160, PG 78), selected in the cDNA library, including a large portion of the nucleocapsid (N), matrix (M) and peplomeric (S) protein genes , were used as probes for a slot blot hybridization assay. Two probe labelling techniques were compared, radiolabeled with [sup 32]P and enzymatic labeled through covalent linkage to peroxidase for chemiluminescence detection. The radioactive probe 174 detected as little as 1-3 pg of viral RNA, while the less sensitive enzymatic probe could not reveal more than 100 pg of RNA. No significant detection amplification was achieved when a mixture of the three probes was used. Probe 174 allowed specific identification for BECV. No hybridization was noticed either with rotaviruses or even with other antigenically unrelated members of the family Coronaviridae such as transmissible gastroenteritis virus. The test proved valid for detection of BECV in the supernatant of infected HRT-18 cells: genomic RNA could be detected after direct spotting of samples, but prior nucleic acid extraction after proteinase K treatment improved virus detection. BECV diagnosis in fecal samples using enzymatic probe was compared with conventional diagnostic methods. (authors).

Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in skin epithelial cells and PDGF mRNA in connective tissue fibroblasts

International Nuclear Information System (INIS)

Antoniades, H.N.; Galanopoulos, T.; Neville-Golden, J.; Kiritsy, C.P.; Lynch, S.E.

1991-01-01

Platelet-derived growth factor (PDGF) stimulates many of the processes important in tissue repair, including proliferation of fibroblasts and synthesis of extracellular matrices. In this study, the authors have demonstrated with in situ hydridization and immunocytochemistry the reversible expression of 3-sis/PDGF-2 and PDGF receptor (PDGF-R) b mRNAs and their respective protein products in epithelial cells and fibroblasts following cutaneous injury in pigs. Epithelial cells in control, unwounded skin did not express c-sis and PDGF-R mRNAs, and fibroblasts expressed only PDGF-R mRNA. The expression levels in the injured site were correlated with the stage of tissue repair, being highest during the initial stages of the repair process and declining at the time of complete re-epithelialization and tissue remodeling. These studies provide a mulecular basis for understanding the mechanisms contributing to normal tissue repair. They suggest the possibility that a defect in these mechanisms may be associated with defective wound healing. It is also conceivable that chronic injury may induce irreversible gene expression leading to pathologic, unregulated cell growth

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5 ' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

DEFF Research Database (Denmark)

Belsham, Graham; Nielsen, Inge; Normann, Preben

2008-01-01

The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 59 untranslated region (5'UTR), contain extensive s...

MERS coronaviruses from camels in Africa exhibit region-dependent genetic diversity.

Science.gov (United States)

Chu, Daniel K W; Hui, Kenrie P Y; Perera, Ranawaka A P M; Miguel, Eve; Niemeyer, Daniela; Zhao, Jincun; Channappanavar, Rudragouda; Dudas, Gytis; Oladipo, Jamiu O; Traoré, Amadou; Fassi-Fihri, Ouafaa; Ali, Abraham; Demissié, Getnet F; Muth, Doreen; Chan, Michael C W; Nicholls, John M; Meyerholz, David K; Kuranga, Sulyman A; Mamo, Gezahegne; Zhou, Ziqi; So, Ray T Y; Hemida, Maged G; Webby, Richard J; Roger, Francois; Rambaut, Andrew; Poon, Leo L M; Perlman, Stanley; Drosten, Christian; Chevalier, Veronique; Peiris, Malik

2018-03-20

Middle East respiratory syndrome coronavirus (MERS-CoV) causes a zoonotic respiratory disease of global public health concern, and dromedary camels are the only proven source of zoonotic infection. Although MERS-CoV infection is ubiquitous in dromedaries across Africa as well as in the Arabian Peninsula, zoonotic disease appears confined to the Arabian Peninsula. MERS-CoVs from Africa have hitherto been poorly studied. We genetically and phenotypically characterized MERS-CoV from dromedaries sampled in Morocco, Burkina Faso, Nigeria, and Ethiopia. Viruses from Africa (clade C) are phylogenetically distinct from contemporary viruses from the Arabian Peninsula (clades A and B) but remain antigenically similar in microneutralization tests. Viruses from West (Nigeria, Burkina Faso) and North (Morocco) Africa form a subclade, C1, that shares clade-defining genetic signatures including deletions in the accessory gene ORF4b Compared with human and camel MERS-CoV from Saudi Arabia, virus isolates from Burkina Faso (BF785) and Nigeria (Nig1657) had lower virus replication competence in Calu-3 cells and in ex vivo cultures of human bronchus and lung. BF785 replicated to lower titer in lungs of human DPP4-transduced mice. A reverse genetics-derived recombinant MERS-CoV (EMC) lacking ORF4b elicited higher type I and III IFN responses than the isogenic EMC virus in Calu-3 cells. However, ORF4b deletions may not be the major determinant of the reduced replication competence of BF785 and Nig1657. Genetic and phenotypic differences in West African viruses may be relevant to zoonotic potential. There is an urgent need for studies of MERS-CoV at the animal-human interface. Copyright © 2018 the Author(s). Published by PNAS.

Mechanisms of Host Receptor Adaptation by Severe Acute Respiratory Syndrome Coronavirus

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Wu, Kailang; Peng, Guiqing; Wilken, Matthew; Geraghty, Robert J.; Li, Fang (UMMC)

2012-12-10

The severe acute respiratory syndrome coronavirus (SARS-CoV) from palm civets has twice evolved the capacity to infect humans by gaining binding affinity for human receptor angiotensin-converting enzyme 2 (ACE2). Numerous mutations have been identified in the receptor-binding domain (RBD) of different SARS-CoV strains isolated from humans or civets. Why these mutations were naturally selected or how SARS-CoV evolved to adapt to different host receptors has been poorly understood, presenting evolutionary and epidemic conundrums. In this study, we investigated the impact of these mutations on receptor recognition, an important determinant of SARS-CoV infection and pathogenesis. Using a combination of biochemical, functional, and crystallographic approaches, we elucidated the molecular and structural mechanisms of each of these naturally selected RBD mutations. These mutations either strengthen favorable interactions or reduce unfavorable interactions with two virus-binding hot spots on ACE2, and by doing so, they enhance viral interactions with either human (hACE2) or civet (cACE2) ACE2. Therefore, these mutations were viral adaptations to either hACE2 or cACE2. To corroborate the above analysis, we designed and characterized two optimized RBDs. The human-optimized RBD contains all of the hACE2-adapted residues (Phe-442, Phe-472, Asn-479, Asp-480, and Thr-487) and possesses exceptionally high affinity for hACE2 but relative low affinity for cACE2. The civet-optimized RBD contains all of the cACE2-adapted residues (Tyr-442, Pro-472, Arg-479, Gly-480, and Thr-487) and possesses exceptionally high affinity for cACE2 and also substantial affinity for hACE2. These results not only illustrate the detailed mechanisms of host receptor adaptation by SARS-CoV but also provide a molecular and structural basis for tracking future SARS-CoV evolution in animals.

Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome.

Directory of Open Access Journals (Sweden)

Albrecht von Brunn

2007-05-01

Full Text Available The severe acute respiratory syndrome coronavirus (SARS-CoV genome is predicted to encode 14 functional open reading frames, leading to the expression of up to 30 structural and non-structural protein products. The functions of a large number of viral ORFs are poorly understood or unknown. In order to gain more insight into functions and modes of action and interaction of the different proteins, we cloned the viral ORFeome and performed a genome-wide analysis for intraviral protein interactions and for intracellular localization. 900 pairwise interactions were tested by yeast-two-hybrid matrix analysis, and more than 65 positive non-redundant interactions, including six self interactions, were identified. About 38% of interactions were subsequently confirmed by CoIP in mammalian cells. Nsp2, nsp8 and ORF9b showed a wide range of interactions with other viral proteins. Nsp8 interacts with replicase proteins nsp2, nsp5, nsp6, nsp7, nsp8, nsp9, nsp12, nsp13 and nsp14, indicating a crucial role as a major player within the replication complex machinery. It was shown by others that nsp8 is essential for viral replication in vitro, whereas nsp2 is not. We show that also accessory protein ORF9b does not play a pivotal role for viral replication, as it can be deleted from the virus displaying normal plaque sizes and growth characteristics in Vero cells. However, it can be expected to be important for the virus-host interplay and for pathogenicity, due to its large number of interactions, by enhancing the global stability of the SARS proteome network, or play some unrealized role in regulating protein-protein interactions. The interactions identified provide valuable material for future studies.

Stochastic theory of interfacial enzyme kinetics: A kinetic Monte Carlo study

International Nuclear Information System (INIS)

Das, Biswajit; Gangopadhyay, Gautam

2012-01-01

Graphical abstract: Stochastic theory of interfacial enzyme kinetics is formulated. Numerical results of macroscopic phenomenon of lag-burst kinetics is obtained by using a kinetic Monte Carlo approach to single enzyme activity. Highlights: ► An enzyme is attached with the fluid state phospholipid molecules on the Langmuir monolayer. ► Through the diffusion, the enzyme molecule reaches the gel–fluid interface. ► After hydrolysing a phospholipid molecule it predominantly leaves the surface in the lag phase. ► The enzyme is strictly attached to the surface with scooting mode of motion and the burst phase appears. - Abstract: In the spirit of Gillespie’s stochastic approach we have formulated a theory to explore the advancement of the interfacial enzyme kinetics at the single enzyme level which is ultimately utilized to obtain the ensemble average macroscopic feature, lag-burst kinetics. We have provided a theory of the transition from the lag phase to the burst phase kinetics by considering the gradual development of electrostatic interaction among the positively charged enzyme and negatively charged product molecules deposited on the phospholipid surface. It is shown that the different diffusion time scales of the enzyme over the fluid and product regions are responsible for the memory effect in the correlation of successive turnover events of the hopping mode in the single trajectory analysis which again is reflected on the non-Gaussian distribution of turnover times on the macroscopic kinetics in the lag phase unlike the burst phase kinetics.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

International Nuclear Information System (INIS)

Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs

Energy Technology Data Exchange (ETDEWEB)

Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Liu, Chao, E-mail: liuchao9@mail.sysu.edu.cn [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Zhang, Hui [Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China); Key Laboratory of Tropical Disease Control of Ministry of Education, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou 510080 (China)

2015-12-15

Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle. - Highlights: • MOV10 can function as a co-factor of HIV-1 Rev. • MOV10 facilitates Rev/RRE-dependent transport of viral mRNAs. • MOV10 interacts with Rev in an RNA-independent manner. • The DEAG-box of MOV10 is required for the enhancement of Rev/RRE-dependent export.

Physical kinetics

International Nuclear Information System (INIS)

Lifschitz, E.M.; Pitajewski, L.P.

1983-01-01

The textbook covers the subject under the following headings: kinetic gas theory, diffusion approximation, collisionless plasma, collisions within the plasma, plasma in the magnetic field, theory of instabilities, dielectrics, quantum fluids, metals, diagram technique for nonequilibrium systems, superconductors, and kinetics of phase transformations

Uncoupling of the hnRNP Npl3p from mRNAs during the stress-induced block in mRNA export.

Science.gov (United States)

Krebber, H; Taura, T; Lee, M S; Silver, P A

1999-08-01

Npl3p, the major mRNA-binding protein of the yeast Saccharomyces cerevisiae shuttles between the nucleus and the cytoplasm. A single amino acid change in the carboxyl terminus of Npl3p (E409 --> K) renders the mutant protein largely cytoplasmic because of a delay in its import into the nucleus. This import defect can be reversed by increasing the intracellular concentration of Mtr10p, the nuclear import receptor for Npl3p. Conversely, using this mutant, we show that Npl3p and mRNA export out of the nucleus is significantly slowed in cells bearing mutations in XPO1/CRM1, which encodes the export receptor for NES-containing proteins and in RAT7, which encodes an essential nucleoporin. Interestingly, following induction of stress by heat shock, high salt, or ethanol, conditions under which most mRNA export is blocked, Npl3p is still exported from the nucleus. The stress-induced export of Npl3p is independent of both the activity of Xpo1p and the continued selective export of heat-shock mRNAs that occurs following stress. UV-cross-linking experiments show that Npl3p is bound to mRNA under normal conditions, but is no longer RNA associated in stressed cells. Taken together, we suggest that the uncoupling of Npl3p and possibly other mRNA-binding proteins from mRNAs in the nucleus provides a general switch that regulates mRNA export. By this model, under normal conditions Npl3p is a major component of an export-competent RNP complex. However, under conditions of stress, Npl3p no longer associates with the export complex, rendering it export incompetent and thus nuclear.

Tracing Airline Travelers for a Public Health Investigation: Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Infection in the United States, 2014.

Science.gov (United States)

Regan, Joanna J; Jungerman, M Robynne; Lippold, Susan A; Washburn, Faith; Roland, Efrosini; Objio, Tina; Schembri, Christopher; Gulati, Reena; Edelson, Paul J; Alvarado-Ramy, Francisco; Pesik, Nicki; Cohen, Nicole J

2016-01-01

CDC routinely conducts contact investigations involving travelers on commercial conveyances, such as aircrafts, cargo vessels, and cruise ships. The agency used established systems of communication and partnerships with other federal agencies to quickly provide accurate traveler contact information to states and jurisdictions to alert contacts of potential exposure to two travelers with Middle East Respiratory Syndrome Coronavirus (MERS-CoV) who had entered the United States on commercial flights in April and May 2014. Applying the same process used to trace and notify travelers during routine investigations, such as those for tuberculosis or measles, CDC was able to notify most travelers of their potential exposure to MERS-CoV during the first few days of each investigation. To prevent the introduction and spread of newly emerging infectious diseases, travelers need to be located and contacted quickly.

Sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction detecting feline coronavirus mutations in effusion and serum/plasma of cats to diagnose feline infectious peritonitis.

Science.gov (United States)

Felten, Sandra; Leutenegger, Christian M; Balzer, Hans-Joerg; Pantchev, Nikola; Matiasek, Kaspar; Wess, Gerhard; Egberink, Herman; Hartmann, Katrin

2017-08-02

Feline coronavirus (FCoV) exists as two pathotypes, and FCoV spike gene mutations are considered responsible for the pathotypic switch in feline infectious peritonitis (FIP) pathogenesis. The aim of this study was to evaluate sensitivity and specificity of a real-time reverse transcriptase polymerase chain reaction (RT-PCR) specifically designed to detect FCoV spike gene mutations at two nucleotide positions. It was hypothesized that this test would correctly discriminate feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV). The study included 63 cats with signs consistent with FIP. FIP was confirmed in 38 cats. Twenty-five control cats were definitively diagnosed with a disease other than FIP. Effusion and/or serum/plasma samples were examined by real-time RT-PCR targeting the two FCoV spike gene fusion peptide mutations M1058 L and S1060A using an allelic discrimination approach. Sensitivity, specificity, negative and positive predictive values including 95% confidence intervals (95% CI) were calculated. FIPV was detected in the effusion of 25/59 cats, one of them being a control cat with chronic kidney disease. A mixed population of FIPV/FECV was detected in the effusion of 2/59 cats; all of them had FIP. RT-PCR was negative or the pathotype could not be determined in 34/59 effusion samples. In effusion, sensitivity was 68.6% (95% CI 50.7-83.2), specificity was 95.8% (95% CI 78.9-99.9). No serum/plasma samples were positive for FIPV. Although specificity of the test in effusions was high, one false positive result occurred. The use of serum/plasma cannot be recommended due to a low viral load in blood.

Identification of two critical amino acid residues of the severe acute respiratory syndrome coronavirus spike protein for its variation in zoonotic tropism transition via a double substitution strategy.

Science.gov (United States)

Qu, Xiu-Xia; Hao, Pei; Song, Xi-Jun; Jiang, Si-Ming; Liu, Yan-Xia; Wang, Pei-Gang; Rao, Xi; Song, Huai-Dong; Wang, Sheng-Yue; Zuo, Yu; Zheng, Ai-Hua; Luo, Min; Wang, Hua-Lin; Deng, Fei; Wang, Han-Zhong; Hu, Zhi-Hong; Ding, Ming-Xiao; Zhao, Guo-Ping; Deng, Hong-Kui

2005-08-19

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a recently identified human coronavirus. The extremely high homology of the viral genomic sequences between the viruses isolated from human (huSARS-CoV) and those of palm civet origin (pcSARS-CoV) suggested possible palm civet-to-human transmission. Genetic analysis revealed that the spike (S) protein of pcSARS-CoV and huSARS-CoV was subjected to the strongest positive selection pressure during transmission, and there were six amino acid residues within the receptor-binding domain of the S protein being potentially important for SARS progression and tropism. Using the single-round infection assay, we found that a two-amino acid substitution (N479K/T487S) of a huSARS-CoV for those of pcSARS-CoV almost abolished its infection of human cells expressing the SARS-CoV receptor ACE2 but no effect upon the infection of mouse ACE2 cells. Although single substitution of these two residues had no effects on the infectivity of huSARS-CoV, these recombinant S proteins bound to human ACE2 with different levels of reduced affinity, and the two-amino acid-substituted S protein showed extremely low affinity. On the contrary, substitution of these two amino acid residues of pcSARS-CoV for those of huSRAS-CoV made pcSARS-CoV capable of infecting human ACE2-expressing cells. These results suggest that amino acid residues at position 479 and 487 of the S protein are important determinants for SARS-CoV tropism and animal-to-human transmission.

Kinetics of phase transformations

International Nuclear Information System (INIS)

Thompson, M.O.; Aziz, M.J.; Stephenson, G.B.

1992-01-01

This volume contains papers presented at the Materials Research Society symposium on Kinetics of Phase Transformations held in Boston, Massachusetts from November 26-29, 1990. The symposium provided a forum for research results in an exceptionally broad and interdisciplinary field. Presentations covered nearly every major class of transformations including solid-solid, liquid-solid, transport phenomena and kinetics modeling. Papers involving amorphous Si, a dominant topic at the symposium, are collected in the first section followed by sections on four major areas of transformation kinetics. The symposium opened with joint sessions on ion and electron beam induced transformations in conjunction with the Surface Chemistry and Beam-Solid Interactions: symposium. Subsequent sessions focused on the areas of ordering and nonlinear diffusion kinetics, solid state reactions and amorphization, kinetics and defects of amorphous silicon, and kinetics of melting and solidification. Seven internationally recognized invited speakers reviewed many of the important problems and recent results in these areas, including defects in amorphous Si, crystal to glass transformations, ordering kinetics, solid-state amorphization, computer modeling, and liquid/solid transformations

Rapid detection of bovine coronavirus by a semi-nested RT-PCR Detecção rápida do Coronavírus Bovino (BCoV por meio de uma semi-nested RT-PCR

Directory of Open Access Journals (Sweden)

Karen M. Asano

2009-11-01

Full Text Available Bovine coronavirus (BCoV is a member of the group 2 of the Coronavirus (Nidovirales: Coronaviridae and the causative agent of enteritis in both calves and adult bovine, as well as respiratory disease in calves. The present study aimed to develop a semi-nested RT-PCR for the detection of BCoV based on representative up-to-date sequences of the nucleocapsid gene, a conserved region of coronavirus genome. Three primers were designed, the first round with a 463bp and the second (semi-nested with a 306bp predicted fragment. The analytical sensitivity was determined by 10-fold serial dilutions of the BCoV Kakegawa strain (HA titre: 256 in DEPC treated ultra-pure water, in fetal bovine serum (FBS and in a BCoV-free fecal suspension, when positive results were found up to the 10-2, 10-3 and 10-7 dilutions, respectively, which suggests that the total amount of RNA in the sample influence the precipitation of pellets by the method of extraction used. When fecal samples was used, a large quantity of total RNA serves as carrier of BCoV RNA, demonstrating a high analytical sensitivity and lack of possible substances inhibiting the PCR. The final semi-nested RT-PCR protocol was applied to 25 fecal samples from adult cows, previously tested by a nested RT-PCR RdRp used as a reference test, resulting in 20 and 17 positives for the first and second tests, respectively, and a substantial agreement was found by kappa statistics (0.694. The high sensitivity and specificity of the new proposed method and the fact that primers were designed based on current BCoV sequences give basis to a more accurate diagnosis of BCoV-caused diseases, as well as to further insights on protocols for the detection of other Coronavirus representatives of both Animal and Public Health importance.O Coronavírus bovino (BCoV pertence ao grupo 2 do gênero Coronavirus (Nidovirales: Coronaviridae e é agente causador de enterites tanto em bezerros como em bovinos adultos, bem como de doen

Kinetics in radiation chemistry

International Nuclear Information System (INIS)

Hummel, A.

1987-01-01

In this chapter the authors first briefly review the kinetics of first- and second-order processes for continuous and pulsed irradiation, without taking the effects of nonhomogeneous formation of the species into consideration. They also discuss diffusion controlled reactions under conditions where interactions of more than two particles can be neglected, first the kinetics of the diffusion-controlled reaction of randomly generated species (homogeneous reaction) and then that of isolated pairs of reactants. The latter is often called geminate kinetics when dealing with pairs of oppositely charged species; they shall use this term for the kinetics of isolated pairs in general. In the last section they discuss briefly the kinetics of groups of more than two reactants

Circulating mRNAs and miRNAs as candidate markers for the diagnosis and prognosis of prostate cancer

DEFF Research Database (Denmark)

Souza, Marilesia Ferreira de; Kuasne, Hellen; Barros-Filho, Mateus de Camargo

2017-01-01

Circulating nucleic acids are found in free form in body fluids and may serve as minimally invasive tools for cancer diagnosis and prognosis. Only a few studies have investigated the potential application of circulating mRNAs and microRNAs (miRNAs) in prostate cancer (PCa). The Cancer Genome Atlas......RNA expression revealed eleven genes and eight miRNAs which were validated by RT-qPCR in plasma samples from 102 untreated PCa patients and 50 cancer-free individuals. Two genes, OR51E2 and SIM2, and two miRNAs, miR-200c and miR-200b, showed significant association with PCa. Expression levels...... of these transcripts distinguished PCa patients from controls (67% sensitivity and 75% specificity). PCa patients and controls with prostate-specific antigen (PSA) ≤ 4.0 ng/mL were discriminated based on OR51E2 and SIM2 expression levels. The miR-200c expression showed association with Gleason score and miR-200b...

Unusually effective microRNA targeting within repeat-rich coding regions of mammalian mRNAs

Science.gov (United States)

Schnall-Levin, Michael; Rissland, Olivia S.; Johnston, Wendy K.; Perrimon, Norbert; Bartel, David P.; Berger, Bonnie

2011-01-01

MicroRNAs (miRNAs) regulate numerous biological processes by base-pairing with target messenger RNAs (mRNAs), primarily through sites in 3′ untranslated regions (UTRs), to direct the repression of these targets. Although miRNAs have sometimes been observed to target genes through sites in open reading frames (ORFs), large-scale studies have shown such targeting to be generally less effective than 3′ UTR targeting. Here, we show that several miRNAs each target significant groups of genes through multiple sites within their coding regions. This ORF targeting, which mediates both predictable and effective repression, arises from highly repeated sequences containing miRNA target sites. We show that such sequence repeats largely arise through evolutionary duplications and occur particularly frequently within families of paralogous C2H2 zinc-finger genes, suggesting the potential for their coordinated regulation. Examples of ORFs targeted by miR-181 include both the well-known tumor suppressor RB1 and RBAK, encoding a C2H2 zinc-finger protein and transcriptional binding partner of RB1. Our results indicate a function for repeat-rich coding sequences in mediating post-transcriptional regulation and reveal circumstances in which miRNA-mediated repression through ORF sites can be reliably predicted. PMID:21685129

Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation.

Science.gov (United States)

Fan, Jun; Zhou, Quan; Li, Yan; Song, Xiuling; Hu, Jijie; Qin, Zaisheng; Tang, Jing; Tao, Tao

2018-01-01

Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq). A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO) analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Profiling of Long Non-coding RNAs and mRNAs by RNA-Sequencing in the Hippocampi of Adult Mice Following Propofol Sedation

Directory of Open Access Journals (Sweden)

Jun Fan

2018-03-01

Full Text Available Propofol is a frequently used intravenous anesthetic agent. The impairment caused by propofol on the neural system, especially the hippocampus, has been widely reported. However, the molecular mechanism underlying the effects of propofol on learning and memory functions in the hippocampus is still unclear. In the present study we performed lncRNA and mRNA analysis in the hippocampi of adult mice, after propofol sedation, through RNA-Sequencing (RNA-Seq. A total of 146 differentially expressed lncRNAs and 1103 mRNAs were identified. Bioinformatics analysis, including gene ontology (GO analysis, pathway analysis and network analysis, were done for the identified dysregulated genes. Pathway analysis indicated that the FoxO signaling pathway played an important role in the effects of propofol on the hippocampus. Finally, four lncRNAs and three proteins were selected from the FoxO-related network for further validation. The up-regulation of lncE230001N04Rik and the down-regulation of lncRP23-430H21.1 and lncB230206L02Rik showed the same fold change tendencies but changes in Gm26532 were not statistically significant in the RNA-Seq results, following propofol sedation. The FoxO pathway-related proteins, PI3K and AKT, are up-regulated in propofol-exposed group. FoxO3a is down-regulated at both mRNA and protein levels. Our study reveals that propofol sedation can influence the expression of lncRNAs and mRNAs in the hippocampus, and bioinformatics analysis have identified key biological processes and pathways associated with propofol sedation. Cumulatively, our results provide a framework for further study on the role of lncRNAs in propofol-induced or -related neurotoxicity, particularly with regards to hippocampus-related dysfunction.

Kinetic equation solution by inverse kinetic method

International Nuclear Information System (INIS)

Salas, G.

1983-01-01

We propose a computer program (CAMU) which permits to solve the inverse kinetic equation. The CAMU code is written in HPL language for a HP 982 A microcomputer with a peripheral interface HP 9876 A ''thermal graphic printer''. The CAMU code solves the inverse kinetic equation by taking as data entry the output of the ionization chambers and integrating the equation with the help of the Simpson method. With this program we calculate the evolution of the reactivity in time for a given disturbance

Introduction to chemical kinetics

CERN Document Server

Soustelle, Michel

2013-01-01

This book is a progressive presentation of kinetics of the chemical reactions. It provides complete coverage of the domain of chemical kinetics, which is necessary for the various future users in the fields of Chemistry, Physical Chemistry, Materials Science, Chemical Engineering, Macromolecular Chemistry and Combustion. It will help them to understand the most sophisticated knowledge of their future job area. Over 15 chapters, this book present the fundamentals of chemical kinetics, its relations with reaction mechanisms and kinetic properties. Two chapters are then devoted to experimental re

Proteome profile of swine testicular cells infected with porcine transmissible gastroenteritis coronavirus.

Directory of Open Access Journals (Sweden)

Ruili Ma

Full Text Available The interactions occurring between a virus and a host cell during a viral infection are complex. The purpose of this paper was to analyze altered cellular protein levels in porcine transmissible gastroenteritis coronavirus (TGEV-infected swine testicular (ST cells in order to determine potential virus-host interactions. A proteomic approach using isobaric tags for relative and absolute quantitation (iTRAQ-coupled two-dimensional liquid chromatography-tandem mass spectrometry identification was conducted on the TGEV-infected ST cells. The results showed that the 4-plex iTRAQ-based quantitative approach identified 4,112 proteins, 146 of which showed significant changes in expression 48 h after infection. At 64 h post infection, 219 of these proteins showed significant change, further indicating that a larger number of proteomic changes appear to occur during the later stages of infection. Gene ontology analysis of the altered proteins showed enrichment in multiple biological processes, including cell adhesion, response to stress, generation of precursor metabolites and energy, cell motility, protein complex assembly, growth, developmental maturation, immune system process, extracellular matrix organization, locomotion, cell-cell signaling, neurological system process, and cell junction organization. Changes in the expression levels of transforming growth factor beta 1 (TGF-β1, caspase-8, and heat shock protein 90 alpha (HSP90α were also verified by western blot analysis. To our knowledge, this study is the first time the response profile of ST host cells following TGEV infection has been analyzed using iTRAQ technology, and our description of the late proteomic changes that are occurring after the time of vigorous viral production are novel. Therefore, this study provides a solid foundation for further investigation, and will likely help us to better understand the mechanisms of TGEV infection and pathogenesis.

Supercritical kinetic analysis in simplified system of fuel debris using integral kinetic model

International Nuclear Information System (INIS)

Tuya, Delgersaikhan; Obara, Toru

2016-01-01

Highlights: • Kinetic analysis in simplified weakly coupled fuel debris system was performed. • The integral kinetic model was used to simulate criticality accidents. • The fission power and released energy during simulated accident were obtained. • Coupling between debris regions and its effect on the fission power was obtained. - Abstract: Preliminary prompt supercritical kinetic analyses in a simplified coupled system of fuel debris designed to roughly resemble a melted core of a nuclear reactor were performed using an integral kinetic model. The integral kinetic model, which can describe region- and time-dependent fission rate in a coupled system of arbitrary geometry, was used because the fuel debris system is weakly coupled in terms of neutronics. The results revealed some important characteristics of coupled systems, such as the coupling between debris regions and the effect of the coupling on the fission rate and released energy in each debris region during the simulated criticality accident. In brief, this study showed that the integral kinetic model can be applied to supercritical kinetic analysis in fuel debris systems and also that it can be a useful tool for investigating the effect of the coupling on consequences of a supercritical accident.

Principles of chemical kinetics

CERN Document Server

House, James E

2007-01-01

James House's revised Principles of Chemical Kinetics provides a clear and logical description of chemical kinetics in a manner unlike any other book of its kind. Clearly written with detailed derivations, the text allows students to move rapidly from theoretical concepts of rates of reaction to concrete applications. Unlike other texts, House presents a balanced treatment of kinetic reactions in gas, solution, and solid states. The entire text has been revised and includes many new sections and an additional chapter on applications of kinetics. The topics covered include quantitative rela

Human trabecular meshwork cells express BMP antagonist mRNAs and proteins.

Science.gov (United States)

Tovar-Vidales, Tara; Fitzgerald, Ashley M; Clark, Abbot F

2016-06-01

Glaucoma patients have elevated aqueous humor and trabecular meshwork (TM) levels of transforming growth factor-beta2 (TGF-β2). TGF-β2 has been associated with increased extracellular matrix (ECM) deposition (i.e. fibronectin), which is attributed to the increased resistance of aqueous humor outflow through the TM. We have previously demonstrated that bone morphogenetic protein (BMP) 4 selectively counteracts the profibrotic effect of TGF-β2 with respect to ECM synthesis in the TM, and this action is reversed by the BMP antagonist gremlin. Thus, the BMP and TGF-β signaling pathways antagonize each other's antifibrotic and profibrotic roles. The purpose of this study was to determine whether cultured human TM cells: (a) express other BMP antagonists including noggin, chordin, BMPER, BAMBI, Smurf1 and 2, and (b) whether expression of these proteins is regulated by exogenous TGF-β2 treatment. Primary human trabecular meshwork (TM) cells were grown to confluency and treated with TGF-β2 (5 ng/ml) for 24 or 48 h in serum-free medium. Untreated cell served as controls. qPCR and Western immunoblots (WB) determined that human TM cells expressed mRNAs and proteins for the BMP antagonist proteins: noggin, chordin, BMPER, BAMBI, and Smurf1/2. Exogenous TGF-β2 decreased chordin, BMPER, BAMBI, and Smurf1 mRNA and protein expression. In contrast, TGF-β2 increased secreted noggin and Smurf2 mRNA and protein levels. BMP antagonist members are expressed in the human TM. These molecules may be involved in the normal function of the TM as well as TM pathogenesis. Altered expression of BMP antagonist members may lead to functional changes in the human TM. Copyright © 2016 Elsevier Ltd. All rights reserved.

Plasma kinetic theory

International Nuclear Information System (INIS)

Elliott, J.A.

1993-01-01

Plasma kinetic theory is discussed and a comparison made with the kinetic theory of gases. The plasma is described by a modified set of fluid equations and it is shown how these fluid equations can be derived. (UK)

Distinct patterns of IFITM-mediated restriction of filoviruses, SARS coronavirus, and influenza A virus.

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I-Chueh Huang

2011-01-01

Full Text Available Interferon-inducible transmembrane proteins 1, 2, and 3 (IFITM1, 2, and 3 are recently identified viral restriction factors that inhibit infection mediated by the influenza A virus (IAV hemagglutinin (HA protein. Here we show that IFITM proteins restricted infection mediated by the entry glycoproteins (GP(1,2 of Marburg and Ebola filoviruses (MARV, EBOV. Consistent with these observations, interferon-β specifically restricted filovirus and IAV entry processes. IFITM proteins also inhibited replication of infectious MARV and EBOV. We observed distinct patterns of IFITM-mediated restriction: compared with IAV, the entry processes of MARV and EBOV were less restricted by IFITM3, but more restricted by IFITM1. Moreover, murine Ifitm5 and 6 did not restrict IAV, but efficiently inhibited filovirus entry. We further demonstrate that replication of infectious SARS coronavirus (SARS-CoV and entry mediated by the SARS-CoV spike (S protein are restricted by IFITM proteins. The profile of IFITM-mediated restriction of SARS-CoV was more similar to that of filoviruses than to IAV. Trypsin treatment of receptor-associated SARS-CoV pseudovirions, which bypasses their dependence on lysosomal cathepsin L, also bypassed IFITM-mediated restriction. However, IFITM proteins did not reduce cellular cathepsin activity or limit access of virions to acidic intracellular compartments. Our data indicate that IFITM-mediated restriction is localized to a late stage in the endocytic pathway. They further show that IFITM proteins differentially restrict the entry of a broad range of enveloped viruses, and modulate cellular tropism independently of viral receptor expression.

Phylogenetic analysis of feline coronavirus strains in an epizootic outbreak of feline infectious peritonitis.

Science.gov (United States)

Barker, E N; Tasker, S; Gruffydd-Jones, T J; Tuplin, C K; Burton, K; Porter, E; Day, M J; Harley, R; Fews, D; Helps, C R; Siddell, S G

2013-01-01

Feline coronavirus (FCoV) infection is common. In a small percentage of cats, FCoV infection is associated with the fatal disease feline infectious peritonitis (FIP). Genetically distinct virulent and avirulent strains of FCoV might coexist within a cat population. To determine whether the strains of FCoV in FIP-affected cats are closely related or genetically distinct from the fecally derived strains of FCoV in contemporary-asymptomatic cats during an epizootic outbreak of FIP. Four cats euthanized because of FIP and 16 asymptomatic cats. This prospective outbreak investigation was initiated during an outbreak of FIP in cats within or rehomed from a rescue/rehoming center. Postmortem samples were collected from cats with FIP and contemporaneous fecal samples from asymptomatic cats. RNA was purified from tissue and fecal samples, FCoV gene fragments were reverse transcribed, PCR-amplified using novel primers, and sequenced. Sequences were aligned with ClustalW and compared with published FCoV sequences. FCoV RNA was detected in all 4 FIP cat postmortem samples and in 9 of the 16 fecal samples from contemporary-asymptomatic cats. Novel primers successfully amplified fragments from 4 regions of the genome for all FCoV-positive samples. Phylogenetic analysis showed that the FIP-associated strains of FCoV from the outbreak were very closely related to the fecally derived strains of FCoV from contemporary-asymptomatic cats. Sequence analysis provided no evidence that genetically distinct virulent and avirulent strains of FCoV were present during this FIP outbreak. Copyright © 2013 by the American College of Veterinary Internal Medicine.

Kinetic Interface

DEFF Research Database (Denmark)

2009-01-01

A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises.......A kinetic interface for orientation detection in a video training system is disclosed. The interface includes a balance platform instrumented with inertial motion sensors. The interface engages a participant's sense of balance in training exercises....

Detection of subgenomic mRNA of feline coronavirus by real-time polymerase chain reaction based on primer-probe energy transfer (P-sg-QPCR)

DEFF Research Database (Denmark)

Hornyák, Ákos; Bálint, Ádám; Farsang, Attila

2012-01-01

Feline infectious peritonitis is one of the most severe devastating diseases of the Felidae. Upon the appearance of clinical signs, a cure for the infected animal is impossible. Therefore rapid and proper diagnosis for both the presence of the causative agent, feline coronavirus (FCo......V) and the manifestation of feline infectious peritonitis is of paramount importance. In the present work, a novel real-time RT-PCR method is described which is able to detect FCoV and to determine simultaneously the quantity of the viral RNA. The new assay combines the M gene subgenomic messenger RNA (sg-mRNA) detection...... assay was proven by positive amplification from a set of nine different FCoV strains and negative from the tested non-coronaviral targets. Examination of faecal samples of healthy young cats, organ samples of perished animals, which suffered from feline infectious peritonitis, and cat leukocytes from...

A pandemic risk assessment of middle east respiratory syndrome coronavirus (MERS-CoV in Saudi Arabia

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Saleh A. Eifan

2017-11-01

Full Text Available Since the initial emergence of Middle East respiratory syndrome coronavirus (MERS-CoV in 2012, a high incidence rate has been observed in Saudi Arabia. This suggests that the country is at continuous risk. The epidemic level of MERS-CoV infection was examined in Saudi Arabia by the Susceptible-Infectious-Recovered (SIR model using a Bayesian approach for estimation of time dependent reproduction number (R across a two-year interval (May, 2013-May, 2015 in five defined clusters, followed by sensitivity analysis of the most significant clusters. Significant MERS-CoV peaks were detected in the period between March and May of each year. Moreover, MERS-CoV infection was highlighted in western (40.8% and central (31.9% regions, followed by eastern region (20%. The temporal-based Bayesian approach indicated a sub-critical epidemic in all regions in the baseline scenario (R: 0.85–0.97. However, R potential limit was exceeded in the sensitivity analysis scenario in only central and western regions (R: 1.08–1.12 that denoted epidemic level in those regions. The impact of sporadic cases was found relatively insignificant and pinpointed to the lack of zoonotic influence on MERS-CoV transmission dynamics. The results of current study would be helpful for evaluation of future progression of MERS-CoV infections, better understanding and control interventions.

Factors Influencing Emergency Nurses' Burnout During an Outbreak of Middle East Respiratory Syndrome Coronavirus in Korea.

Science.gov (United States)

Kim, Ji Soo; Choi, Jeong Sil

2016-12-01

Emergency department (ED) nurses suffer from persistent stress after experiencing the traumatic event of exposure to Middle East respiratory syndrome coronavirus (MERS-CoV), which can subsequently lead to burnout. This study aimed to assess ED nurses' burnout level during an outbreak of MERS-CoV and to identify influencing factors in order to provide basic information for lowering and preventing the level of burnout. Study participants were ED nurses working in eight hospitals designated for treating MERS-CoV-infected patients in Korea. We performed multiple regression analysis to explore the factors influencing burnout. The ED nurses' burnout was affected by job stress (β=0.59, pburnout. ED nurses taking care of MERS-CoV-infected patients should be aware that burnout is higher for nurses in their divisions than nurses in other hospital departments and that job stress is the biggest influential factor of burnout. To be ready for the outbreak of emerging contagious diseases such as MERS-CoV, efforts and preparations should be made to reduce burnout. Job stress should be managed and resolved. Working conditions for mitigating job stress and systematic stress management programs should be provided, and hospital resources for the treatment of MERS-CoV need to be reinforced. Moreover, promoting support from family and friends is required. Copyright © 2016. Published by Elsevier B.V.

Kinetics and

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Mojtaba Ahmadi

2016-11-01

Full Text Available The aqueous degradation of Reactive Yellow 84 (RY84 by potassium peroxydisulfate (K2S2O8 has been studied in laboratory scale experiments. The effect of the initial concentrations of potassium peroxydisulfate and RY84, pH and temperature on RY84 degradation were also examined. Experimental data were analyzed using first and second-order kinetics. The degradation kinetics of RY84 of the potassium peroxydisulfate process followed the second-order reaction kinetics. These rate constants have an extreme values similar to of 9.493 mM−1min−1 at a peroxydisulfate dose of 4 mmol/L. Thermodynamic parameters such as activation (Ea and Gibbs free energy (ΔG° were also evaluated. The negative value of ΔGo and Ea shows the spontaneous reaction natural conditions and exothermic nature.

Time-Dependent Expression Profiles of microRNAs and mRNAs in Rat Milk Whey

Science.gov (United States)

Izumi, Hirohisa; Kosaka, Nobuyoshi; Shimizu, Takashi; Sekine, Kazunori; Ochiya, Takahiro; Takase, Mitsunori

2014-01-01

Functional RNAs, such as microRNA (miRNA) and mRNA, are present in milk, but their roles are unknown. To clarify the roles of milk RNAs, further studies using experimental animals such as rats are needed. However, it is unclear whether rat milk also contains functional RNAs and what their time dependent expression profiles are. Thus, we prepared total RNA from whey isolated from rat milk collected on days 2, 9, and 16 postpartum and analyzed using microarrays and quantitative PCR. The concentration of RNA in colostrum whey (day 2) was markedly higher than that in mature milk whey (days 9 and 16). Microarray analysis detected 161 miRNAs and 10,948 mRNA transcripts. Most of the miRNAs and mRNA transcripts were common to all tested milks. Finally, we selected some immune- and development-related miRNAs and mRNAs, and analysed them by quantitative PCR (in equal sample volumes) to determine their time-dependent changes in expression in detail. Some were significantly more highly expressed in colostrum whey than in mature milk whey, but some were expressed equally. And mRNA expression levels of some cytokines and hormones did not reflect the protein levels. It is still unknown whether RNAs in milk play biological roles in neonates. However, our data will help guide future in vivo studies using experimental animals such as rats. PMID:24533154

Slow VO2 off-kinetics in skeletal muscle is associated with fast PCr off-kinetics--and inversely.

Science.gov (United States)

Korzeniewski, Bernard; Zoladz, Jerzy A

2013-09-01

The computer model of the bioenergetic system in skeletal muscle, developed previously, was used to study the effect of the characteristic decay time of the parallel activation of oxidative phosphorylation [τ(OFF)] during muscle recovery on the muscle oxygen consumption rate (Vo2) and phosphocreatine (PCr) work-to-rest transition (off)-kinetics and on the relationship between the Vo2 and PCr rest-to-work transition (on)- and off-kinetics in moderate and heavy exercise. An increase in τ(OFF) slows down the initial phase of the muscle Vo2 off-kinetics and accelerates the PCr off-kinetics. As a result, the relationship between the initial phase of the Vo2 off-kinetics (lasting approximately 3-60 s in computer simulations) and the PCr off-kinetics is inverse: the slower the former, the faster the latter. A faster initial phase of the Vo2 off-kinetics is associated with a slower late phase of the Vo2 off-kinetics, and as a result, the integral of Vo2 above baseline during recovery, representing the oxygen debt, is identical in all cases [values of τ(OFF)] for a given PCr decrease. Depending on τ(OFF), the muscle Vo2 on-kinetics was either equally fast or slower than the Vo2 off-kinetics in moderate exercise and always slower in heavy exercise. PCr on-kinetics was always faster than PCr off-kinetics. This study clearly demonstrates that τ(OFF) has a pronounced impact on the mutual relations between the muscle Vo2 and PCr on- and off-kinetics.

Holographic kinetic k-essence model

Energy Technology Data Exchange (ETDEWEB)

Cruz, Norman [Departamento de Fisica, Facultad de Ciencia, Universidad de Santiago de Chile, Casilla 307, Santiago (Chile)], E-mail: ncruz@lauca.usach.cl; Gonzalez-Diaz, Pedro F.; Rozas-Fernandez, Alberto [Colina de los Chopos, Instituto de Fisica Fundamental, Consejo Superior de Investigaciones Cientificas, Serrano 121, 28006 Madrid (Spain)], E-mail: a.rozas@cfmac.csic.es; Sanchez, Guillermo [Departamento de Matematica y Ciencia de la Computacion, Facultad de Ciencia, Universidad de Santiago de Chile, Casilla 307, Santiago (Chile)], E-mail: gsanchez@usach.cl

2009-08-31

We consider a connection between the holographic dark energy density and the kinetic k-essence energy density in a flat FRW universe. With the choice c{>=}1, the holographic dark energy can be described by a kinetic k-essence scalar field in a certain way. In this Letter we show this kinetic k-essential description of the holographic dark energy with c{>=}1 and reconstruct the kinetic k-essence function F(X)

Reflected kinetics model for nuclear space reactor kinetics and control scoping calculations

Energy Technology Data Exchange (ETDEWEB)

Washington, K.E.

1986-05-01

The objective of this research is to develop a model that offers an alternative to the point kinetics (PK) modelling approach in the analysis of space reactor kinetics and control studies. Modelling effort will focus on the explicit treatment of control drums as reactivity input devices so that the transition to automatic control can be smoothly done. The proposed model is developed for the specific integration of automatic control and the solution of the servo mechanism problem. The integration of the kinetics model with an automatic controller will provide a useful tool for performing space reactor scoping studies for different designs and configurations. Such a tool should prove to be invaluable in the design phase of a space nuclear system from the point of view of kinetics and control limitations.

Reflected kinetics model for nuclear space reactor kinetics and control scoping calculations

International Nuclear Information System (INIS)

Washington, K.E.

1986-05-01

The objective of this research is to develop a model that offers an alternative to the point kinetics (PK) modelling approach in the analysis of space reactor kinetics and control studies. Modelling effort will focus on the explicit treatment of control drums as reactivity input devices so that the transition to automatic control can be smoothly done. The proposed model is developed for the specific integration of automatic control and the solution of the servo mechanism problem. The integration of the kinetics model with an automatic controller will provide a useful tool for performing space reactor scoping studies for different designs and configurations. Such a tool should prove to be invaluable in the design phase of a space nuclear system from the point of view of kinetics and control limitations

Transcription pattern of UL131A-128 mRNA in clinical strains of ...

Indian Academy of Sciences (India)

Human cytomegalovirus (HCMV) mRNA was obtained from human embryonic lung fibroblast cells infected by HCMV clinical strains from urine samples of infants at different kinetic periods. The cDNA of UL131A-128 mRNAs was amplified using reverse transcription-polymerase chain reaction (RT-PCR) and analysed by ...

LLNL Chemical Kinetics Modeling Group

Energy Technology Data Exchange (ETDEWEB)

Pitz, W J; Westbrook, C K; Mehl, M; Herbinet, O; Curran, H J; Silke, E J

2008-09-24

The LLNL chemical kinetics modeling group has been responsible for much progress in the development of chemical kinetic models for practical fuels. The group began its work in the early 1970s, developing chemical kinetic models for methane, ethane, ethanol and halogenated inhibitors. Most recently, it has been developing chemical kinetic models for large n-alkanes, cycloalkanes, hexenes, and large methyl esters. These component models are needed to represent gasoline, diesel, jet, and oil-sand-derived fuels.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP

Science.gov (United States)

Wickramasinghe, Vihandha O.; Andrews, Robert; Ellis, Peter; Langford, Cordelia; Gurdon, John B.; Stewart, Murray; Venkitaraman, Ashok R.; Laskey, Ronald A.

2014-01-01

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression. PMID:24510098

Gyrocenter-gauge kinetic theory

International Nuclear Information System (INIS)

Qin, H.; Tang, W.M.; Lee, W.W.

2000-01-01

Gyrocenter-gauge kinetic theory is developed as an extension of the existing gyrokinetic theories. In essence, the formalism introduced here is a kinetic description of magnetized plasmas in the gyrocenter coordinates which is fully equivalent to the Vlasov-Maxwell system in the particle coordinates. In particular, provided the gyroradius is smaller than the scale-length of the magnetic field, it can treat high frequency range as well as the usual low frequency range normally associated with gyrokinetic approaches. A significant advantage of this formalism is that it enables the direct particle-in-cell simulations of compressional Alfven waves for MHD applications and of RF waves relevant to plasma heating in space and laboratory plasmas. The gyrocenter-gauge kinetic susceptibility for arbitrary wavelength and arbitrary frequency electromagnetic perturbations in a homogeneous magnetized plasma is shown to recover exactly the classical result obtained by integrating the Vlasov-Maxwell system in the particle coordinates. This demonstrates that all the waves supported by the Vlasov-Maxwell system can be studied using the gyrocenter-gauge kinetic model in the gyrocenter coordinates. This theoretical approach is so named to distinguish it from the existing gyrokinetic theory, which has been successfully developed and applied to many important low-frequency and long parallel wavelength problems, where the conventional meaning of gyrokinetic has been standardized. Besides the usual gyrokinetic distribution function, the gyrocenter-gauge kinetic theory emphasizes as well the gyrocenter-gauge distribution function, which sometimes contains all the physics of the problems being studied, and whose importance has not been realized previously. The gyrocenter-gauge distribution function enters Maxwell's equations through the pull-back transformation of the gyrocenter transformation, which depends on the perturbed fields. The efficacy of the gyrocenter-gauge kinetic approach is

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia.

Science.gov (United States)

Klahre, U; Hemmings-Mieszczak, M; Filipowicz, W

1995-06-01

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3'-untranslated regions (3'-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3' non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event occurring in this intron.

Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus.

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Taisheng Li

2006-12-01

Full Text Available Most of the individuals infected with SARS coronavirus (SARS-CoV spontaneously recovered without clinical intervention. However, the immunological correlates associated with patients' recovery are currently unknown. In this report, we have sequentially monitored 30 recovered patients over a two-year period to characterize temporal changes in SARS-CoV-specific antibody responses as well as cytotoxic T cell (CTL responses. We have found persistence of robust antibody and CTL responses in all of the study subjects throughout the study period, with a moderate decline one year after the onset of symptoms. We have also identified two potential major CTL epitopes in N proteins based on ELISPOT analysis of pooled peptides. However, despite the potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients have not yet been restored to normal levels. In summary, our study has, for the first time, characterized the temporal and dynamic changes of humoral and CTL responses in the natural history of SARS-recovered individuals, and strongly supports the notion that high and sustainable levels of immune responses correlate strongly with the disease outcome. Our findings have direct implications for future design and development of effective therapeutic agents and vaccines against SARS-CoV infection.

Discrepancies between feline coronavirus antibody and nucleic acid detection in effusions of cats with suspected feline infectious peritonitis.

Science.gov (United States)

Lorusso, Eleonora; Mari, Viviana; Losurdo, Michele; Lanave, Gianvito; Trotta, Adriana; Dowgier, Giulia; Colaianni, Maria Loredana; Zatelli, Andrea; Elia, Gabriella; Buonavoglia, Domenico; Decaro, Nicola

2017-10-31

Intra-vitam diagnosis of feline infectious peritonitis (FIP) is a challenge for veterinary diagnosticians, since there are no highly specific and sensitive assays currently available. With the aim to contribute to fill this diagnostic gap, a total of 61 effusions from cats with suspected effusive FIP were collected intra-vitam for detection of feline coronavirus (FCoV) antibodies and RNA by means of indirect immunofluorescence (IIF) assay and real-time RT-PCR (qRT-PCR), respectively. In 5 effusions there was no evidence for either FCoV RNA or antibodies, 51 and 52 specimens tested positive by IIF and qRT-PCR, respectively, although antibody titres≥1:1600, which are considered highly suggestive of FIP, were detected only in 37 effusions. Three samples with high antibody levels tested negative by qRT-PCR, whereas 18 qRT-PCR positive effusions contained no or low-titre antibodies. qRT-PCR positive samples with low antibody titres mostly contained low FCoV RNA loads, although the highest antibody titres were detected in effusions with C T values>30. In conclusion, combining the two methods, i.e., antibody and RNA detection would help improving the intra-vitam diagnosis of effusive FIP. Copyright © 2017 Elsevier Ltd. All rights reserved.

Proteomic analysis of chicken embryonic trachea and kidney tissues after infection in ovo by avian infectious bronchitis coronavirus

Directory of Open Access Journals (Sweden)

Kong Xiangang

2011-03-01

Full Text Available Abstract Background Avian infectious bronchitis (IB is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV. Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS. Results 17 differentially expressed proteins from tracheal tissues and 19 differentially expressed proteins from kidney tissues were identified. These proteins mostly related to the cytoskeleton, binding of calcium ions, the stress response, anti-oxidative, and macromolecular metabolism. Some of these altered proteins were confirmed further at the mRNA level using real-time RT-PCR. Moreover, western blotting analysis further confirmed the changes of annexin A5 and HSPB1 during IBV infection. Conclusions To the best of our knowledge, we have performed the first analysis of the proteomic changes in chicken embryonic trachea and kidney tissues during IBV infection in ovo. The data obtained should facilitate a better understanding of the pathogenesis of IBV infection.

BST2/CD317 counteracts human coronavirus 229E productive infection by tethering virions at the cell surface

Energy Technology Data Exchange (ETDEWEB)

Wang, Shiu-Mei [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China); Huang, Kuo-Jung [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Wang, Chin-Tien, E-mail: chintien@ym.edu.tw [Department of Medical Research and Education, Taipei Veterans General Hospital and Institute of Clinical Medicine, Taipei 11217, Taiwan (China); Institute of Clinical Medicine, National Yang-Ming University School of Medicine, Taipei, Taiwan (China)

2014-01-20

Bone marrow stromal antigen 2 (BST2), an interferon-inducible antiviral factor, has been shown to block the release of various enveloped viruses from cells. It has also been identified as an innate immune system component. Most enveloped viruses subject to BST2 restriction bud at the plasma membrane. Here we report our findings that (a) the production of human coronavirus 229E (HCoV-229E) progeny viruses, whose budding occurs at the ER-Golgi intermediate compartment (ERGIC), markedly decreases in the presence of BST2; and (b) BST2 knockdown expression results in enhanced HCoV-229E virion production. Electron microscopy analyses indicate that HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. Our results suggest that BST2 exerts a broad blocking effect against enveloped virus release, regardless of whether budding occurs at the plasma membrane or intracellular compartments. - Highlights: • BST2 knockdown expression results in enhanced HCoV-229E egress. • HCoV-229E virions are tethered to cell surfaces or intracellular membranes by BST2. • HCoV-229E infection at high MOI can significantly downregulate HeLa BST2 and rescue HIV-1 egress.

Kinetics and hybrid kinetic-fluid models for nonequilibrium gas and plasmas

International Nuclear Information System (INIS)

Crouseilles, N.

2004-12-01

For a few decades, the application of the physics of plasmas has appeared in different fields like laser-matter interaction, astrophysics or thermonuclear fusion. In this thesis, we are interested in the modeling and the numerical study of nonequilibrium gas and plasmas. To describe such systems, two ways are usually used: the fluid description and the kinetic description. When we study a nonequilibrium system, fluid models are not sufficient and a kinetic description have to be used. However, solving a kinetic model requires the discretization of a large number of variables, which is quite expensive from a numerical point of view. The aim of this work is to propose a hybrid kinetic-fluid model thanks to a domain decomposition method in the velocity space. The derivation of the hybrid model is done in two different contexts: the rarefied gas context and the more complicated plasmas context. The derivation partly relies on Levermore's entropy minimization approach. The so-obtained model is then discretized and validated on various numerical test cases. In a second stage, a numerical study of a fully kinetic model is presented. A collisional plasma constituted of electrons and ions is considered through the Vlasov-Poisson-Fokker-Planck-Landau equation. Then, a numerical scheme which preserves total mass and total energy is presented. This discretization permits in particular a numerical study of the Landau damping. (author)

Tantalum high-temperature oxidation kinetics

International Nuclear Information System (INIS)

Grigor'ev, Yu.M.; Sarkisyan, A.A.; Merzhanov, A.G.

1981-01-01

Kinetics of heat release and scale growth during tantalum oxidation within 650-1300 deg C temperature range in oxygen-containing media is investigated. Kinetic equations and temperature and pressure dependences of constants are ound Applicability of the kinetic Lorie mechanism for the description of the tantalum oxidation kinetics applicably to rapid-passing processes is shown. It is stated that the process rate (reaction ability) is determined by adsorption desorption factors on the external surface of the ''protective'' oxide for the ''linear'' oxidation stage [ru

Muscular Oxygen Uptake Kinetics in Aged Adults.

Science.gov (United States)

Koschate, J; Drescher, U; Baum, K; Eichberg, S; Schiffer, T; Latsch, J; Brixius, K; Hoffmann, U

2016-06-01

Pulmonary oxygen uptake (V˙O2) kinetics and heart rate kinetics are influenced by age and fitness. Muscular V˙O2 kinetics can be estimated from heart rate and pulmonary V˙O2. In this study the applicability of a test using pseudo-random binary sequences in combination with a model to estimate muscular V˙O2 kinetics was tested. Muscular V˙O2 kinetics were expected to be faster than pulmonary V˙O2 kinetics, slowed in aged subjects and correlated with maximum V˙O2 and heart rate kinetics. 27 elderly subjects (73±3 years; 81.1±8.2 kg; 175±4.7 cm) participated. Cardiorespiratory kinetics were assessed using the maximum of cross-correlation functions, higher maxima implying faster kinetics. Muscular V˙O2 kinetics were faster than pulmonary V˙O2 kinetics (0.31±0.1 vs. 0.29±0.1 s; p=0.004). Heart rate kinetics were not correlated with muscular or pulmonary V˙O2 kinetics or maximum V˙O2. Muscular V˙O2 kinetics correlated with maximum V˙O2 (r=0.35; p=0.033). This suggests, that muscular V˙O2 kinetics are faster than estimates from pulmonary V˙O2 and related to maximum V˙O2 in aged subjects. In the future this experimental approach may help to characterize alterations in muscular V˙O2 under various conditions independent of motivation and maximal effort. © Georg Thieme Verlag KG Stuttgart · New York.

pp32 (ANP32A expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs.

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Timothy K Williams

Full Text Available The expression of protein phosphatase 32 (PP32, ANP32A is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1, a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C, but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK, causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR, while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.

Adsorption analysis equilibria and kinetics

CERN Document Server

Do, Duong D

1998-01-01

This book covers topics of equilibria and kinetics of adsorption in porous media. Fundamental equilibria and kinetics are dealt with for homogeneous as well as heterogeneous particles. Five chapters of the book deal with equilibria and eight chapters deal with kinetics. Single component as well as multicomponent systems are discussed. In kinetics analysis, we deal with the various mass transport processes and their interactions inside a porous particle. Conventional approaches as well as the new approach using Maxwell-Stefan equations are presented. Various methods to measure diffusivity, such

Multi-Organ Damage in Human Dipeptidyl Peptidase 4 Transgenic Mice Infected with Middle East Respiratory Syndrome-Coronavirus.

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Guangyu Zhao

Full Text Available The Middle East Respiratory Syndrome Coronavirus (MERS-CoV causes severe acute respiratory failure and considerable extrapumonary organ dysfuction with substantial high mortality. For the limited number of autopsy reports, small animal models are urgently needed to study the mechanisms of MERS-CoV infection and pathogenesis of the disease and to evaluate the efficacy of therapeutics against MERS-CoV infection. In this study, we developed a transgenic mouse model globally expressing codon-optimized human dipeptidyl peptidase 4 (hDPP4, the receptor for MERS-CoV. After intranasal inoculation with MERS-CoV, the mice rapidly developed severe pneumonia and multi-organ damage, with viral replication being detected in the lungs on day 5 and in the lungs, kidneys and brains on day 9 post-infection. In addition, the mice exhibited systemic inflammation with mild to severe pneumonia accompanied by the injury of liver, kidney and spleen with neutrophil and macrophage infiltration. Importantly, the mice exhibited symptoms of paralysis with high viral burden and viral positive neurons on day 9. Taken together, this study characterizes the tropism of MERS-CoV upon infection. Importantly, this hDPP4-expressing transgenic mouse model will be applicable for studying the pathogenesis of MERS-CoV infection and investigating the efficacy of vaccines and antiviral agents designed to combat MERS-CoV infection.

Antisense sequences and antagomiR 155 in therapy for B lymphomas over expressing miR-155: preclinical models and identification of target mRNAs

International Nuclear Information System (INIS)

Marziali, G.; Peschle, C.

2009-01-01

Micro RNAs (miRNAs) are a conserved class of small noncoding RNAs (22-25 nucleotides), which modulate gene expression at post-transcriptional level by base pairing to the 3'UTR of the target mRNAs, thus causing messenger degradation or inhibiting its translation. Experimental evidence indicate that several miRNAs are deregulated in human tumors. MIRN155 has been shown to be highly expressed in a variety of human B cell lymphomas, especially diffuse large B cells, Hodgkin, and a subset of Burkitt lymphomas. Its expression is physiologically increased in activated B and T cells and it plays a key role in regulating the homeostasis and function of the immune system

Ezrin interacts with the SARS coronavirus Spike protein and restrains infection at the entry stage.

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Jean Kaoru Millet

Full Text Available BACKGROUND: Entry of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV and its envelope fusion with host cell membrane are controlled by a series of complex molecular mechanisms, largely dependent on the viral envelope glycoprotein Spike (S. There are still many unknowns on the implication of cellular factors that regulate the entry process. METHODOLOGY/PRINCIPAL FINDINGS: We performed a yeast two-hybrid screen using as bait the carboxy-terminal endodomain of S, which faces the cytosol during and after opening of the fusion pore at early stages of the virus life cycle. Here we show that the ezrin membrane-actin linker interacts with S endodomain through the F1 lobe of its FERM domain and that both the eight carboxy-terminal amino-acids and a membrane-proximal cysteine cluster of S endodomain are important for this interaction in vitro. Interestingly, we found that ezrin is present at the site of entry of S-pseudotyped lentiviral particles in Vero E6 cells. Targeting ezrin function by small interfering RNA increased S-mediated entry of pseudotyped particles in epithelial cells. Furthermore, deletion of the eight carboxy-terminal amino acids of S enhanced S-pseudotyped particles infection. Expression of the ezrin dominant negative FERM domain enhanced cell susceptibility to infection by SARS-CoV and S-pseudotyped particles and potentiated S-dependent membrane fusion. CONCLUSIONS/SIGNIFICANCE: Ezrin interacts with SARS-CoV S endodomain and limits virus entry and fusion. Our data present a novel mechanism involving a cellular factor in the regulation of S-dependent early events of infection.

Molecular and phylogenetic characterization of bovine coronavirus virus isolated from dairy cattle in Central Region, Thailand.

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Singasa, Kanokwan; Songserm, Taweesak; Lertwatcharasarakul, Preeda; Arunvipas, Pipat

2017-10-01

Bovine coronavirus (BCoV) is involved mainly in enteric infections in cattle. This study reports the first molecular detection of BCoV in a diarrhea outbreak in dairy cows in the Central Region, Thailand. BCoV was molecularly detected from bloody diarrheic cattle feces by using nested PCR. Agarose gel electrophoresis of three diarrheic fecal samples yielded from the 25 samples desired amplicons that were 488 base pairs and sequencing substantiated that have BCoV. The sequence alignment indicated that nucleotide and amino acid sequences, the three TWD isolated in Thailand, were more quite homologous to each other (amino acid at position 39 of TWD1, TWD3 was proline, but TWD2 was serine) and closely related to OK-0514-3strain (virulent respiratory strain; RBCoV).The amino acid sequencing identities among TWD1, TWD2,TWD3, and OK-0514-3 strain were 96.0 to 96.6%, those at which T3I, H65N, D87G, H127Y, andQ136R were changed. In addition, the phylogenetic tree of the hypervariable region S1subunit spike glycoprotein BCoV gene was composed of three major clades by using the 54 sequences generated and showed that the evolutionally distance, TWD1, TWD2, and TWD3 were the isolated group together and most similar to OK-0514-3 strain (98.2 to 98.5% similarity). Further study will develop ELISA assay for serologic detection of winter dysentery disease.

Present status on numerical algorithms and benchmark tests for point kinetics and quasi-static approximate kinetics

International Nuclear Information System (INIS)

Ise, Takeharu

1976-12-01

Review studies have been made on algorithms of numerical analysis and benchmark tests on point kinetics and quasistatic approximate kinetics computer codes to perform efficiently benchmark tests on space-dependent neutron kinetics codes. Point kinetics methods have now been improved since they can be directly applied to the factorization procedures. Methods based on Pade rational function give numerically stable solutions and methods on matrix-splitting are interested in the fact that they are applicable to the direct integration methods. An improved quasistatic (IQ) approximation is the best and the most practical method; it is numerically shown that the IQ method has a high stability and precision and the computation time which is about one tenth of that of the direct method. IQ method is applicable to thermal reactors as well as fast reactors and especially fitted for fast reactors to which many time steps are necessary. Two-dimensional diffusion kinetics codes are most practicable though there exist also three-dimensional diffusion kinetics code as well as two-dimensional transport kinetics code. On developing a space-dependent kinetics code, in any case, it is desirable to improve the method so as to have a high computing speed for solving static diffusion and transport equations. (auth.)

Granulocyte kinetics

International Nuclear Information System (INIS)

Peters, A.M.; Lavender, J.P.; Saverymuttu, S.H.

1985-01-01

By using density gradient materials enriched with autologous plasma, the authors have been able to isolate granulocutes from other cellular elements and label them with In-111 without separation from a plasma environment. The kinetic behavior of these cells suggests that phenomena attributed to granulocyte activation are greatly reduced by this labeling. Here, they review their study of granulocyte kinetics in health and disease in hope of quantifying sites of margination and identifying principal sites of destruction. The three principle headings of the paper are distribution, life-span, and destruction

ME31B globally represses maternal mRNAs by two distinct mechanisms during the Drosophila maternal-to-zygotic transition.

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Wang, Miranda; Ly, Michael; Lugowski, Andrew; Laver, John D; Lipshitz, Howard D; Smibert, Craig A; Rissland, Olivia S

2017-09-06

In animal embryos, control of development is passed from exclusively maternal gene products to those encoded by the embryonic genome in a process referred to as the maternal-to-zygotic transition (MZT). We show that the RNA-binding protein, ME31B, binds to and represses the expression of thousands of maternal mRNAs during the Drosophila MZT. However, ME31B carries out repression in different ways during different phases of the MZT. Early, it represses translation while, later, its binding leads to mRNA destruction, most likely as a consequence of translational repression in the context of robust mRNA decay. In a process dependent on the PNG kinase, levels of ME31B and its partners, Cup and Trailer Hitch (TRAL), decrease by over 10-fold during the MZT, leading to a change in the composition of mRNA-protein complexes. We propose that ME31B is a global repressor whose regulatory impact changes based on its biological context.

Irreversible processes kinetic theory

CERN Document Server

Brush, Stephen G

2013-01-01

Kinetic Theory, Volume 2: Irreversible Processes deals with the kinetic theory of gases and the irreversible processes they undergo. It includes the two papers by James Clerk Maxwell and Ludwig Boltzmann in which the basic equations for transport processes in gases are formulated, together with the first derivation of Boltzmann's ""H-theorem"" and a discussion of this theorem, along with the problem of irreversibility.Comprised of 10 chapters, this volume begins with an introduction to the fundamental nature of heat and of gases, along with Boltzmann's work on the kinetic theory of gases and s

Drug-Target Kinetics in Drug Discovery.

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Tonge, Peter J

2018-01-17

The development of therapies for the treatment of neurological cancer faces a number of major challenges including the synthesis of small molecule agents that can penetrate the blood-brain barrier (BBB). Given the likelihood that in many cases drug exposure will be lower in the CNS than in systemic circulation, it follows that strategies should be employed that can sustain target engagement at low drug concentration. Time dependent target occupancy is a function of both the drug and target concentration as well as the thermodynamic and kinetic parameters that describe the binding reaction coordinate, and sustained target occupancy can be achieved through structural modifications that increase target (re)binding and/or that decrease the rate of drug dissociation. The discovery and deployment of compounds with optimized kinetic effects requires information on the structure-kinetic relationships that modulate the kinetics of binding, and the molecular factors that control the translation of drug-target kinetics to time-dependent drug activity in the disease state. This Review first introduces the potential benefits of drug-target kinetics, such as the ability to delineate both thermodynamic and kinetic selectivity, and then describes factors, such as target vulnerability, that impact the utility of kinetic selectivity. The Review concludes with a description of a mechanistic PK/PD model that integrates drug-target kinetics into predictions of drug activity.

Physisorption kinetics

CERN Document Server

Kreuzer, Hans Jürgen

1986-01-01

This monograph deals with the kinetics of adsorption and desorption of molecules physisorbed on solid surfaces. Although frequent and detailed reference is made to experiment, it is mainly concerned with the theory of the subject. In this, we have attempted to present a unified picture based on the master equation approach. Physisorption kinetics is by no means a closed and mature subject; rather, in writing this monograph we intended to survey a field very much in flux, to assess its achievements so far, and to give a reasonable basis from which further developments can take off. For this reason we have included many papers in the bibliography that are not referred to in the text but are of relevance to physisorption. To keep this monograph to a reasonable size, and also to allow for some unity in the presentation of the material, we had to omit a number of topics related to physisorption kinetics. We have not covered to any extent the equilibrium properties of physisorbed layers such as structures, phase tr...

A kinetics database and scripts for PHREEQC

Science.gov (United States)

Hu, B.; Zhang, Y.; Teng, Y.; Zhu, C.

2017-12-01

Kinetics of geochemical reactions has been increasingly used in numerical models to simulate coupled flow, mass transport, and chemical reactions. However, the kinetic data are scattered in the literature. To assemble a kinetic dataset for a modeling project is an intimidating task for most. In order to facilitate the application of kinetics in geochemical modeling, we assembled kinetics parameters into a database for the geochemical simulation program, PHREEQC (version 3.0). Kinetics data were collected from the literature. Our database includes kinetic data for over 70 minerals. The rate equations are also programmed into scripts with the Basic language. Using the new kinetic database, we simulated reaction path during the albite dissolution process using various rate equations in the literature. The simulation results with three different rate equations gave difference reaction paths at different time scale. Another application involves a coupled reactive transport model simulating the advancement of an acid plume in an acid mine drainage site associated with Bear Creek Uranium tailings pond. Geochemical reactions including calcite, gypsum, and illite were simulated with PHREEQC using the new kinetic database. The simulation results successfully demonstrated the utility of new kinetic database.

Some Aspects of Extended Kinetic Equation

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Dilip Kumar

2015-09-01

Full Text Available Motivated by the pathway model of Mathai introduced in 2005 [Linear Algebra and Its Applications, 396, 317–328] we extend the standard kinetic equations. Connection of the extended kinetic equation with fractional calculus operator is established. The solution of the general form of the fractional kinetic equation is obtained through Laplace transform. The results for the standard kinetic equation are obtained as the limiting case.

Expression Levels of Myostatin and Matrix Metalloproteinase 14 mRNAs in Uterine Leiomyoma are Correlated With Dysmenorrhea.

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Tsigkou, Anastasia; Reis, Fernando M; Ciarmela, Pasquapina; Lee, Meng H; Jiang, Bingjie; Tosti, Claudia; Shen, Fang-Rong; Shi, Zhendan; Chen, You-Guo; Petraglia, Felice

2015-12-01

Uterine leiomyoma is the most common benign neoplasm of female reproductive system, found in about 50% of women in reproductive age. The mechanisms of leiomyoma growth include cell proliferation, which is modulated by growth factors, and deposition of extracellular matrix (ECM). Activin A and myostatin are growth factors that play a role in proliferation of leiomyoma cells. Matrix metalloproteinases (MMPs) are known for their ability to remodel the ECM in different biological systems. The aim of this study was to evaluate the expression levels of activin βA-subunit, myostatin, and MMP14 messenger RNAs (mRNAs) in uterine leiomyomas and the possible correlation of these factors with clinical features of the disease. Matrix metalloproteinase 14 was highly expressed in uterine leiomyoma and correlated with myostatin and activin A mRNA expression. Moreover, MMP14 and myostatin mRNA expression correlated significantly and directly with the intensity of dysmenorrhea. Overall, the present findings showed that MMP14 mRNA is highly expressed in uterine leiomyoma, where it correlates with the molecular expression of growth factors and is further increased in cases of intense dysmenorrhea. © The Author(s) 2015.

Testosterone stimulates progesterone production and STAR, P450 cholesterol side-chain cleavage and LH receptor mRNAs expression in hen (Gallus domesticus) granulosa cells.

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Rangel, P L; Rodríguez, A; Rojas, S; Sharp, P J; Gutierrez, C G

2009-12-01

The chicken ovary is organized into a hierarchy of yellow yolky follicles that ovulate on successive days. Active or passive immunization of laying hens against testosterone blocks ovulation without affecting follicle development. Testosterone may play a role in pre-ovulatory follicle maturation by stimulating granulosa progesterone production. We assessed whether this stimulus is dose-related and depends on the maturity of the donor follicle, and if it does so by stimulating granulosa cell STAR, P450 cholesterol side-chain cleavage (P450scc), and LH receptor (LHCGR) mRNAs expression. Progesterone production by granulosa cells from F1, F3, and F4 follicles, cultured for 3 h without testosterone was greater in cells collected 11-14 h than 1-4 h after ovulation. These differences in progesterone production were less pronounced after granulosa cells had been cultured for 24 h. Culture of granulosa cells for 3 or 24 h with testosterone (1-100 ng/ml) stimulated progesterone production in cells collected from F4, F3, or F1 follicles 1-4, or 11-14 h after ovulation. Testosterone (0-4000 ng/ml) alone or in combination with LH (0-100 ng/ml) increased progesterone production by F1 granulosa cells, collected 1-4 and 11-14 h after ovulation and cultured for 3 h. Finally, testosterone (10 or 100 ng/ml) increased STAR, P450scc, and LHCGR mRNAs, when added to 3 h cultures of F1 granulosa cells. In conclusion, testosterone stimulates granulosa cell progesterone production in hen pre-ovulatory hierarchical follicles irrespective of maturational state, acting alone or additively with LH. We propose that testosterone promotes granulosa cell maturation to facilitate the pre-ovulatory release of LH.

Vlasov simulations of kinetic Alfvén waves at proton kinetic scales

Energy Technology Data Exchange (ETDEWEB)

Vásconez, C. L. [Dipartimento di Fisica, Università della Calabria, I-87036 Cosenza (Italy); Observatorio Astronómico de Quito, Escuela Politécnica Nacional, Quito (Ecuador); Valentini, F.; Veltri, P. [Dipartimento di Fisica, Università della Calabria, I-87036 Cosenza (Italy); Camporeale, E. [Centrum Wiskunde and Informatica, Amsterdam (Netherlands)

2014-11-15

Kinetic Alfvén waves represent an important subject in space plasma physics, since they are thought to play a crucial role in the development of the turbulent energy cascade in the solar wind plasma at short wavelengths (of the order of the proton gyro radius ρ{sub p} and/or inertial length d{sub p} and beyond). A full understanding of the physical mechanisms which govern the kinetic plasma dynamics at these scales can provide important clues on the problem of the turbulent dissipation and heating in collisionless systems. In this paper, hybrid Vlasov-Maxwell simulations are employed to analyze in detail the features of the kinetic Alfvén waves at proton kinetic scales, in typical conditions of the solar wind environment (proton plasma beta β{sub p} = 1). In particular, linear and nonlinear regimes of propagation of these fluctuations have been investigated in a single-wave situation, focusing on the physical processes of collisionless Landau damping and wave-particle resonant interaction. Interestingly, since for wavelengths close to d{sub p} and β{sub p} ≃ 1 (for which ρ{sub p} ≃ d{sub p}) the kinetic Alfvén waves have small phase speed compared to the proton thermal velocity, wave-particle interaction processes produce significant deformations in the core of the particle velocity distribution, appearing as phase space vortices and resulting in flat-top velocity profiles. Moreover, as the Eulerian hybrid Vlasov-Maxwell algorithm allows for a clean almost noise-free description of the velocity space, three-dimensional plots of the proton velocity distribution help to emphasize how the plasma departs from the Maxwellian configuration of thermodynamic equilibrium due to nonlinear kinetic effects.

Chemical kinetics and reaction mechanism

International Nuclear Information System (INIS)

Jung, Ou Sik; Park, Youn Yeol

1996-12-01

This book is about chemical kinetics and reaction mechanism. It consists of eleven chapters, which deal with reaction and reaction speed on reaction mechanism, simple reaction by rate expression, reversible reaction and simultaneous reaction, successive reaction, complicated reaction mechanism, assumption for reaction mechanism, transition state theory, successive reaction and oscillating reaction, reaction by solution, research method high except kinetics on reaction mechanism, high reaction of kinetics like pulsed radiolysis.

Kinetic modelling of enzymatic starch hydrolysis

NARCIS (Netherlands)

Bednarska, K.A.

2015-01-01

Kinetic modelling of enzymatic starch hydrolysis – a summary

K.A. Bednarska

The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch.

Limitations of using feline coronavirus spike protein gene mutations to diagnose feline infectious peritonitis.

Science.gov (United States)

Barker, Emily N; Stranieri, Angelica; Helps, Chris R; Porter, Emily L; Davidson, Andrew D; Day, Michael J; Knowles, Toby; Kipar, Anja; Tasker, Séverine

2017-10-05

Feline infectious peritonitis (FIP) is a fatal disease of cats, and a sequela of systemic feline coronavirus (FCoV) infection. Mutations in the viral spike (S) gene have been associated with FCoVs found in tissues from cats with FIP, but not FCoVs found in faeces from healthy cats, and are implicated in monocyte/macrophage tropism and systemic spread. This study was designed to determine whether S gene mutation analysis can reliably diagnose FIP. Cats were categorised as with FIP (n = 57) or without FIP (n = 45) based on gross post-mortem and histopathological examination including immunohistochemistry for FCoV antigen. RNA was purified from available tissue, fluid and faeces. Reverse-transcriptase quantitative-PCR (RT-qPCR) was performed on all samples using FCoV-specific primers, followed by sequencing of a section of the S gene on RT-qPCR positive samples. Samples were available from a total of 102 cats. Tissue, fluid, and faecal samples from cats with FIP were more likely to be FCoV RT-qPCR-positive (90.4, 78.4 and 64.6% respectively) than those from cats without FIP (7.8, 2.1 and 20% respectively). Identification of S gene mutated FCoVs as an additional step to the detection of FCoV alone, only moderately increased specificity for tissue samples (from 92.6 to 94.6%) but specificity was unchanged for fluid samples (97.9%) for FIP diagnosis; however, sensitivity was markedly decreased for tissue (from 89.8 to 80.9%) and fluid samples (from 78.4 to 60%) for FIP diagnosis. These findings demonstrate that S gene mutation analysis in FCoVs does not substantially improve the ability to diagnose FIP as compared to detection of FCoV alone.

A critical look at the kinetic models of thermoluminescence-II. Non-first order kinetics

International Nuclear Information System (INIS)

Sunta, C M; Ayta, W E F; Chubaci, J F D; Watanabe, S

2005-01-01

Non-first order (FO) kinetics models are of three types; second order (SO), general order (GO) and mixed order (MO). It is shown that all three of these have constraints in their energy level schemes and their applicable parameter values. In nature such restrictions are not expected to exist. The thermoluminescence (TL) glow peaks produced by these models shift their position and change their shape as the trap occupancies change. Such characteristics are very unlike those found in samples of real materials. In these models, in general, retrapping predominates over recombination. It is shown that the quasi-equilibrium (QE) assumption implied in the derivation of the TL equation of these models is quite valid, thus disproving earlier workers' conclusion that QE cannot be held under retrapping dominant conditions. However notwithstanding their validity, they suffer from the shortcomings as stated above and have certain lacunae. For example, the kinetic order (KO) parameter and the pre-exponential factor which are assumed to be the constant parameters of the GO kinetics expression turn out to be variables when this expression is applied to plausible physical models. Further, in glow peak characterization using the GO expression, the quality of fit is found to deteriorate when the best fitted value of KO parameter is different from 1 and 2. This means that the found value of the basic parameter, namely the activation energy, becomes subject to error. In the MO kinetics model, the value of the KO parameter α would change with dose, and thus in this model also, as in the GO model, no single value of KO can be assigned to a given glow peak. The paper discusses TL of real materials having characteristics typically like those of FO kinetics. Theoretically too, a plausible physical model of TL emission produces glow peaks which have characteristics of FO kinetics under a wide variety of parametric combinations. In the background of the above findings, it is suggested that

Fluorescence kinetics and positron annihilation kinetics investigations in cadmium sulfide crystals

Energy Technology Data Exchange (ETDEWEB)

Grillot, E; Bancie-Grillot, M; Egee, M [Reims Univ., 51 (France)

1976-03-01

Fluorescence kinetics and positrons annihilation kinetics investigations on CdS crystals, either very pure or with increasing contents of Ag-ions, led to similar and complementary results. Ag-ions mainly fill the cadmium vacancies of the lattice, building red emission luminogene centres, while green 'edge-emission' ones are destroyed. These latter, which involve an excited level active for high energy series fluorescence, seems actually related to cadmium vacancies.

Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

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Aline S. Hora

2013-01-01

Full Text Available To evaluate the most controversial issue concerning current feline coronavirus (FCoV virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV in regard to the pathogenesis of feline infectious peritonitis (FIP, this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3 from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

Identification and tissue distribution of mRNAs encoding salmon-type calcitonins-IV and -V in the rainbow trout.

Science.gov (United States)

Hidaka, Yoshie; Suzuki, Masakazu

2004-06-01

Four types of calcitonin are produced in salmonid fish, although their functional diversity is almost unknown. To explore the significance of these isoforms, we have characterized salmon-type calcitonin (sCT) mRNAs in the rainbow trout (Oncorhynchus mykiss), and examined their tissue distribution. In addition to the previously isolated sCT-I cDNAs, two new forms of sCT cDNA were cloned from the ultimobranchial gland, and one of them (sCT-IV cDNA) was predicted to encode an N-terminal peptide of 80 amino acid residues, a putative cleavage site Lys-Arg, sCT-IV, a cleavage and amidation sequence Gly-Lys-Lys-Arg, and a C-terminal peptide of 18 amino acids. The sCT-IV precursor was 78% identical with the rainbow trout sCT-I precursors. The other cloned cDNA encoded a precursor for a novel CT, sCT-V. The sCT-V peptide was different from sCT-IV by only one amino acid residue: Val at position 8 in the latter was replaced by Met. The sCT-V precursor had 80 and 90% identity with the sCT-I and -IV precursors respectively. No cDNA clones were obtained for sCTs-II or -III.Tissue distribution of sCT-I, -IV and -V mRNAs was examined by RT-PCR and specific cleavage with restriction enzymes. An amplified fragment from sCT-I mRNA was detected not only in the ultimobranchial gland, but also in the gills, testis and ovary. RT-PCR analysis coupled to restriction digestion further revealed that sCT-IV mRNA was expressed in both the testis and the ultimobranchial gland. The expression sites of sCT-IV mRNA were localized to the Leydig cells of the testis and to the parenchymal cells of the ultimobranchial gland, by in situ hybridization histochemistry. Although the amino acid sequence of sCT-V peptide was nearly the same as that of sCT-IV, the sCT-V gene showed a much wider pattern of expression: the band amplified by RT-PCR was detected in all the tissues examined except the kidney, gills and blood cells. The sCT-V mRNA was shown to be localized in the parenchymal cells of the

SARS-CoV related Betacoronavirus and diverse Alphacoronavirus members found in western old-world.

Science.gov (United States)

Ar Gouilh, Meriadeg; Puechmaille, Sébastien J; Diancourt, Laure; Vandenbogaert, Mathias; Serra-Cobo, Jordi; Lopez Roïg, Marc; Brown, Paul; Moutou, François; Caro, Valérie; Vabret, Astrid; Manuguerra, Jean-Claude

2018-04-01

The emergence of SARS-CoV and MERS-CoV, triggered the discovery of a high diversity of coronaviruses in bats. Studies from Europe have shown that coronaviruses circulate in bats in France but this reflects only a fraction of the whole diversity. In the current study the diversity of coronaviruses circulating in western Europe was extensively explored. Ten alphacoronaviruses in eleven bat species belonging to the Miniopteridae, Vespertilionidae and Rhinolophidae families and, a SARS-CoV-related Betacoronavirus in Rhinolophus ferrumequinum were identified. The diversity and prevalence of bat coronaviruses presently reported from western Europe is much higher than previously described and includes a SARS-CoV sister group. This diversity demonstrates the dynamic evolution and circulation of coronaviruses in this species. That said, the identified coronaviruses were consistently associated with a particular bat species or genus, and these relationships were maintained no matter the geographic location. The observed phylogenetic grouping of coronaviruses from the same species in Europe and Asia, emphasizes the role of host/pathogen coevolution in this group. Copyright © 2018 Elsevier Inc. All rights reserved.

New Methods for Processing and Quantifying VO2 Kinetics to Steady State: VO2 Onset Kinetics

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Craig R. McNulty

2017-09-01

Full Text Available Current methods of oxygen uptake (VO2 kinetics data handling may be too simplistic for the complex physiology involved in the underlying physiological processes. Therefore, the aim of this study was to quantify the VO2 kinetics to steady state across the full range of sub-ventilatory threshold work rates, with a particular focus on the VO2 onset kinetics. Ten healthy, moderately trained males participated in five bouts of cycling. Each bout involved 10 min at a percentage of the subject's ventilation threshold (30, 45, 60, 75, 90% from unloaded cycling. The VO2 kinetics was quantified using the conventional mono-exponential time constant (tau, τ, as well as the new methods for VO2 onset kinetics. Compared to linear modeling, non-linear modeling caused a deterioration of goodness of fit (main effect, p < 0.001 across all exercise intensities. Remainder kinetics were also improved using a modified application of the mono-exponential model (main effect, p < 0.001. Interestingly, the slope from the linear regression of the onset kinetics data is similar across all subjects and absolute exercise intensities, and thereby independent of subject fitness and τ. This could indicate that there are no functional limitations between subjects during this onset phase, with limitations occurring for the latter transition to steady state. Finally, the continuing use of mono-exponential modeling could mask important underlying physiology of more instantaneous VO2 responses to steady state. Consequently, further research should be conducted on this new approach to VO2 onset kinetics.

Molecular dynamics of Middle East Respiratory Syndrome Coronavirus (MERS CoV) fusion heptad repeat trimers

KAUST Repository

Kandeel, Mahmoud

2018-05-17

Structural studies related to Middle East Respiratory Syndrome Coronavirus (MERS CoV) infection process are so limited. In this study, molecular dynamics (MD) simulation was carried out to unravel changes in the MERS CoV heptad repeat domains (HRs) and factors affecting fusion state HR stability. Results indicated that HR trimer is more rapidly stabilized, having stable system energy and lowest root mean square deviations (RMSDs). While trimers were the predominant active form of CoVs HR, monomers were also discovered in both of viral and cellular membranes. In order to find the differences between S2 monomer and trimer molecular dynamics, S2 monomer were modelled and subjected to MD simulation. In contrast to S2 trimer, S2 monomer was unstable, having high RMSDs with major drifts above 8 Å. Fluctuation of HR residue positions revealed major changes in the C-terminal of HR2 and the linker coil between HR1 and HR2 in both monomer and trimer. Hydrophobic residues at the “a” and “d” positions of HR helices stabilize the whole system, having minimal changes in RMSD. The global distance test and contact area difference scores support instability of MERS CoV S2 monomer. Analysis of HR1-HR2 inter-residue contacts and interaction energy revealed three different energy scales along HR helices. Two strong interaction energies were identified at the start of the HR2 helix and at the C-terminal of HR2. The identified critical residues by MD simulation and residues at a and d position of HR helix were strong stabilizers of HRs recognition.

Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

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Juan Reguera

Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

Evaluation of antibodies against feline coronavirus 7b protein for diagnosis of feline infectious peritonitis in cats.

Science.gov (United States)

Kennedy, Melissa A; Abd-Eldaim, Mohamed; Zika, Sarah E; Mankin, Joseph M; Kania, Stephen A

2008-09-01

To determine whether expression of feline coronavirus (FCoV) 7b protein, as indicated by the presence of specific serum antibodies, consistently correlated with occurrence of feline infectious peritonitis (FIP) in cats. 95 serum samples submitted for various diagnostic assays and 20 samples from specific-pathogen-free cats tested as negative control samples. The 7b gene from a virulent strain of FCoV was cloned into a protein expression vector. The resultant recombinant protein was produced and used in antibody detection assays via western blot analysis of serum samples. Results were compared with those of an immunofluorescence assay (IFA) for FCoV-specific antibody and correlated with health status. Healthy IFA-seronegative cats were seronegative for antibodies against the 7b protein. Some healthy cats with detectable FCoV-specific antibodies as determined via IFA were seronegative for antibodies against the 7b protein. Serum from cats with FIP had antibodies against the 7b protein, including cats with negative results via conventional IFA. However, some healthy cats, as well as cats with conditions other than FIP that were seropositive to FCoV via IFA, were also seropositive for the 7b protein. Expression of the 7b protein, as indicated by detection of antibodies against the protein, was found in most FCoV-infected cats. Seropositivity for this protein was not specific for the FCoV virulent biotype or a diagnosis of FIP.

A retrospective clinical and epidemiological study on feline coronavirus (FCoV) in cats in Istanbul, Turkey.

Science.gov (United States)

Tekelioglu, B K; Berriatua, E; Turan, N; Helps, C R; Kocak, M; Yilmaz, H

2015-04-01

The presence of antibodies to feline coronavirus (FCoV) and feline immunodeficiency virus (FIV), together with feline leukemia virus (FeLV) antigen was investigated in 169 ill household and stray cats attending a veterinary surgery in Istanbul in 2009-14. The estimated FCoV and FIV seroprevalence (95% confidence intervals) were 37% (30-45%) and 11% (6-16%), respectively and FeLV prevalence was 1% (0-3%). FCoV seroprevalence increased until 2 years of age, was highest in 2014 and among household cats living with other cats and with outdoor access, and was lower in FIV seropositive compared to seronegative cats. Symptoms typically associated with wet feline infectious peritonitis (FIP) including ascites, abdominal distention or pleural effusion, coupled in many cases with non-antibiotic responsive fever, were observed in 19% (32/169) of cats, and 75% (24/32) of these cats were FCoV seropositive. FCoV seropositivity was also associated with a high white blood cell count, high plasma globulin, low plasma albumin and low blood urea nitrogen. The percentage of FCoV seropositive and seronegative cats that died in spite of supportive veterinary treatment was 33% (21/63) and 12% (13/106), respectively. These results indicate that FCoV is widespread and has a severe clinical impact in cats from Istanbul. Moreover, the incidence of FCoV infections could be rising, and in the absence of effective vaccination cat owners need to be made aware of ways to minimize the spread of this virus. Copyright © 2015 Elsevier B.V. All rights reserved.

Sink limitation induces the expression of multiple soybean vegetative lipoxygenase mRNAs while the endogenous jasmonic acid level remains low.

Science.gov (United States)

Bunker, T W; Koetje, D S; Stephenson, L C; Creelman, R A; Mullet, J E; Grimes, H D

1995-08-01

The response of individual members of the lipoxygenase multigene family in soybeans to sink deprivation was analyzed. RNase protection assays indicated that a novel vegetative lipoxygenase gene, vlxC, and three other vegetative lipoxygenase mRNAs accumulated in mature leaves in response to a variety of sink limitations. These data suggest that several members of the lipoxygenase multigene family are involved in assimilate partitioning. The possible involvement of jasmonic acid as a signaling molecule regulating assimilate partitioning into the vegetative storage proteins and lipoxygenases was directly assessed by determining the endogenous level of jasmonic acid in leaves from plants with their pods removed. There was no rise in the level of endogenous jasmonic acid coincident with the strong increase in both vlxC and vegetative storage protein VspB transcripts in response to sink limitation. Thus, expression of the vegetative lipoxygenases and vegetative storage proteins is not regulated by jasmonic acid in sink-limited leaves.

Saffman-Taylor fingers with kinetic undercooling

KAUST Repository

Gardiner, Bennett P. J.

2015-02-23

© 2015 American Physical Society. The mathematical model of a steadily propagating Saffman-Taylor finger in a Hele-Shaw channel has applications to two-dimensional interacting streamer discharges which are aligned in a periodic array. In the streamer context, the relevant regularization on the interface is not provided by surface tension but instead has been postulated to involve a mechanism equivalent to kinetic undercooling, which acts to penalize high velocities and prevent blow-up of the unregularized solution. Previous asymptotic results for the Hele-Shaw finger problem with kinetic undercooling suggest that for a given value of the kinetic undercooling parameter, there is a discrete set of possible finger shapes, each analytic at the nose and occupying a different fraction of the channel width. In the limit in which the kinetic undercooling parameter vanishes, the fraction for each family approaches 1/2, suggesting that this "selection" of 1/2 by kinetic undercooling is qualitatively similar to the well-known analog with surface tension. We treat the numerical problem of computing these Saffman-Taylor fingers with kinetic undercooling, which turns out to be more subtle than the analog with surface tension, since kinetic undercooling permits finger shapes which are corner-free but not analytic. We provide numerical evidence for the selection mechanism by setting up a problem with both kinetic undercooling and surface tension and numerically taking the limit that the surface tension vanishes.

Saffman-Taylor fingers with kinetic undercooling

KAUST Repository

Gardiner, Bennett P. J.; McCue, Scott W.; Dallaston, Michael C.; Moroney, Timothy J.

2015-01-01

© 2015 American Physical Society. The mathematical model of a steadily propagating Saffman-Taylor finger in a Hele-Shaw channel has applications to two-dimensional interacting streamer discharges which are aligned in a periodic array. In the streamer context, the relevant regularization on the interface is not provided by surface tension but instead has been postulated to involve a mechanism equivalent to kinetic undercooling, which acts to penalize high velocities and prevent blow-up of the unregularized solution. Previous asymptotic results for the Hele-Shaw finger problem with kinetic undercooling suggest that for a given value of the kinetic undercooling parameter, there is a discrete set of possible finger shapes, each analytic at the nose and occupying a different fraction of the channel width. In the limit in which the kinetic undercooling parameter vanishes, the fraction for each family approaches 1/2, suggesting that this "selection" of 1/2 by kinetic undercooling is qualitatively similar to the well-known analog with surface tension. We treat the numerical problem of computing these Saffman-Taylor fingers with kinetic undercooling, which turns out to be more subtle than the analog with surface tension, since kinetic undercooling permits finger shapes which are corner-free but not analytic. We provide numerical evidence for the selection mechanism by setting up a problem with both kinetic undercooling and surface tension and numerically taking the limit that the surface tension vanishes.

Mechanism for Controlling the Dimer-Monomer Switch and Coupling Dimerization to Catalysis of the Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease

Energy Technology Data Exchange (ETDEWEB)

Shi,J.; Sivaraman, J.; Song, J.

2008-01-01

Unlike 3C protease, the severe acute respiratory syndrome coronavirus (SARS-CoV) 3C-like protease (3CLpro) is only enzymatically active as a homodimer and its catalysis is under extensive regulation by the unique extra domain. Despite intense studies, two puzzles still remain: (i) how the dimer-monomer switch is controlled and (ii) why dimerization is absolutely required for catalysis. Here we report the monomeric crystal structure of the SARS-CoV 3CLpro mutant R298A at a resolution of 1.75 Angstroms . Detailed analysis reveals that Arg298 serves as a key component for maintaining dimerization, and consequently, its mutation will trigger a cooperative switch from a dimer to a monomer. The monomeric enzyme is irreversibly inactivated because its catalytic machinery is frozen in the collapsed state, characteristic of the formation of a short 310-helix from an active-site loop. Remarkably, dimerization appears to be coupled to catalysis in 3CLpro through the use of overlapped residues for two networks, one for dimerization and another for the catalysis.

The gut microbiome and mucosal defenses in cats with coronaviruses: a pilot study

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Sara Meazzi

2017-05-01

Full Text Available Feline Infectious Peritonitis (FIP develops from a mutation of enteric feline coronaviruses (FCoVs and an imbalance of the host immune response. The wide polymorphism of FCoVs is associated with the viral replication rate (Licitra et al. 2013.  Changes in the composition of the gut microbiota may induce quali-quantitative modifications in FCoVs and/or different immune profiles (Weese et al., 2015. Few information is available on feline gut microbiome and the association between microbiota and the predisposition to pathological conditions (Ramadan et al., 2014. The aim of this study is to provide preliminary data about the composition of gut microbiota in healthy cats compared with FCoV infected cats (with and without  FIP, in order to evaluate whether changes of gut microbiota may induce changes in FCoV, in its genetic polymorphism and in the mucosal immunity. Screening analyses have been performed on 22 cats: - Routine hematology and biochemistry on EDTA and serum (included electrophoresis and alpha-1-acid glycoprotein measurement for cats suspected with FIP - Nested RT-PCR-3’UTR on frozen faeces - Effusion evaluation - FIV/FeLV serology Due to strict inclusion criteria (cats younger than 2.5 years old, indoor and not assuming antibiotics in the previous two months and based on the results obtained from the complete set of analysis, only 15 cats, specifically 5 cats for each of the following 3 groups: FIP- affected, healthy negative and positive for FCoV, have been recruited to perform the following analyses:  - microbiota analysis through NGS of 16S rRNA gene (V4 region amplicons followed by bioinformatic analysis  -  evaluation of secretory IgA (ELISA kit - phylogenetic analysis of FCoVs S gene sequences Innovative results will be provided on the feline gut microbiota composition. These will be correlated with the presence and genetic polymorphisms of FCoV and mucosal defenses to establish significant correlations between the analysed

A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice.

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Anjeanette Roberts

2007-01-01

Full Text Available No single animal model for severe acute respiratory syndrome (SARS reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15 that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15, duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as

Chemical kinetics of gas reactions

CERN Document Server

Kondrat'Ev, V N

2013-01-01

Chemical Kinetics of Gas Reactions explores the advances in gas kinetics and thermal, photochemical, electrical discharge, and radiation chemical reactions. This book is composed of 10 chapters, and begins with the presentation of general kinetic rules for simple and complex chemical reactions. The next chapters deal with the experimental methods for evaluating chemical reaction mechanisms and some theories of elementary chemical processes. These topics are followed by discussions on certain class of chemical reactions, including unimolecular, bimolecular, and termolecular reactions. The rema

Relativistic Chiral Kinetic Theory

International Nuclear Information System (INIS)

Stephanov, Mikhail

2016-01-01

This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

Relativistic Chiral Kinetic Theory

Energy Technology Data Exchange (ETDEWEB)

Stephanov, Mikhail

2016-12-15

This very brief review of the recent progress in chiral kinetic theory is based on the results of Refs. [J.-Y. Chen, D. T. Son, M. A. Stephanov, H.-U. Yee, Y. Yin, Lorentz Invariance in Chiral Kinetic Theory, Phys. Rev. Lett. 113 (18) (2014) 182302. doi: (10.1103/PhysRevLett.113.182302); J.-Y. Chen, D. T. Son, M. A. Stephanov, Collisions in Chiral Kinetic Theory, Phys. Rev. Lett. 115 (2) (2015) 021601. doi: (10.1103/PhysRevLett.115.021601); M. A. Stephanov, H.-U. Yee, The no-drag frame for anomalous chiral fluid, Phys. Rev. Lett. 116 (12) (2016) 122302. doi: (10.1103/PhysRevLett.116.122302)].

Kinetic theory of gases and plasmas

International Nuclear Information System (INIS)

Schram, P.P.J.M.

1991-01-01

Kinetic theory provides the link between the non-equilibrium statistical mechanics of many-particle systems and macroscopic or phenomenological physics. This volume deals with the derivation of kinetic equations, their limitations and generalizations,and with the applications of kinetic theory to physical phenomena and the calculation of transport coefficients. This book is divided in 12 chapters which discuss a wide range of topics such as balanced equations, the Klimontovich, Vlasov-Maxwell, and Boltzmann equations, Chapman-Enskog theory, the kinetic theory of plasmas, B.G.K. models, linear response theory, Brownian motion and renormalized kinetic theory. Each chapter is concluded with exercises, which not only enable the readers to test their understanding of the theory, but also present additional examples which complement the text. 151 refs.; 35 figs.; 5 tabs

Crystallization Kinetics within a Generic Modelling Framework

DEFF Research Database (Denmark)

Meisler, Kresten Troelstrup; von Solms, Nicolas; Gernaey, Krist

2013-01-01

An existing generic modelling framework has been expanded with tools for kinetic model analysis. The analysis of kinetics is carried out within the framework where kinetic constitutive models are collected, analysed and utilized for the simulation of crystallization operations. A modelling...... procedure is proposed to gain the information of crystallization operation kinetic model analysis and utilize this for faster evaluation of crystallization operations....

Maternal mRNAs of PEM and macho 1, the ascidian muscle determinant, associate and move with a rough endoplasmic reticulum network in the egg cortex.

Science.gov (United States)

Sardet, Christian; Nishida, Hiroki; Prodon, Francois; Sawada, Kaichiro

2003-12-01

Localization of maternal mRNAs in the egg cortex is an essential feature of polarity in embryos of Drosophila, Xenopus and ascidians. In ascidians, maternal mRNAs such as macho 1, a determinant of primary muscle-cell fate, belong to a class of postplasmic RNAs that are located along the animal-vegetal gradient in the egg cortex. Between fertilization and cleavage, these postplasmic RNAs relocate in two main phases. They further concentrate and segregate in small posterior blastomeres into a cortical structure, the centrosome-attracting body (CAB), which is responsible for unequal cleavages. By using high-resolution, fluorescent, in situ hybridization in eggs, zygotes and embryos of Halocynthia roretzi, we showed that macho 1 and HrPEM are localized on a reticulated structure situated within 2 mum of the surface of the unfertilized egg, and within 8 mum of the surface the vegetal region and then posterior region of the zygote. By isolating cortices from eggs and zygotes we demonstrated that this reticulated structure is a network of cortical rough endoplasmic reticulum (cER) that is tethered to the plasma membrane. The postplasmic RNAs macho 1 and HrPEM were located on the cER network and could be detached from it. We also show that macho 1 and HrPEM accumulated in the CAB and the cER network. We propose that these postplasmic RNAs relocalized after fertilization by following the microfilament- and microtubule-driven translocations of the cER network to the poles of the zygote. We also suggest that the RNAs segregate and concentrate in posterior blastomeres through compaction of the cER to form the CAB. A multimedia BioClip 'Polarity inside the egg cortex' tells the story and can be downloaded at www.bioclips.com/bioclip.html

Selective increases of AMPA, NMDA and kainate receptor subunit mRNAs in the hippocampus and orbitofrontal cortex but not in prefrontal cortex of human alcoholics

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Zhe eJin

2014-01-01

Full Text Available Glutamate is the main excitatory transmitter in the human brain. Drugs that affect the glutamatergic signaling will alter neuronal excitability. Ethanol inhibits glutamate receptors. We examined the expression level of glutamate receptor subunit mRNAs in human post-mortem samples from alcoholics and compared the results to brain samples from control subjects. RNA from hippocampal dentate gyrus (HP-DG, orbitofrontal cortex (OFC, and dorso-lateral prefrontal cortex (DL-PFC samples from 21 controls and 19 individuals with chronic alcohol dependence were included in the study. Total RNA was assayed using quantitative RT-PCR. Out of the 16 glutamate receptor subunits, mRNAs encoding two AMPA (2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-ylpropanoic acid receptor subunits GluA2 and GluA3; three kainate receptor subunits GluK2, GluK3 and GluK5 and five NMDA (N-methyl-D-aspartate receptor subunits GluN1, GluN2A, GluN2C, GluN2D and GluN3A were significantly increased in the HP-DG region in alcoholics. In the OFC, mRNA encoding the NMDA receptor subunit GluN3A was increased, whereas in the DL-PFC, no differences in mRNA levels were observed. Our laboratory has previously shown that the expression of genes encoding inhibitory GABA-A receptors is altered in the HP-DG and OFC of alcoholics (Jin et al., 2011. Whether the changes in one neurotransmitter system drives changes in the other or if they change independently is currently not known. The results demonstrate that excessive long-term alcohol consumption is associated with altered expression of genes encoding glutamate receptors in a brain region-specific manner. It is an intriguing possibility that genetic predisposition to alcoholism may contribute to these gene expression changes.

Experimental feline enteric coronavirus infection reveals an aberrant infection pattern and shedding of mutants with impaired infectivity in enterocyte cultures

Science.gov (United States)

Desmarets, Lowiese M. B.; Vermeulen, Ben L.; Theuns, Sebastiaan; Conceição-Neto, Nádia; Zeller, Mark; Roukaerts, Inge D. M.; Acar, Delphine D.; Olyslaegers, Dominique A. J.; Van Ranst, Marc; Matthijnssens, Jelle; Nauwynck, Hans J.

2016-01-01

Feline infectious peritonitis (FIP) results from mutations in the viral genome during a common feline enteric coronavirus (FECV) infection. Since many virological and immunological data on FECV infections are lacking, the present study investigated these missing links during experimental infection of three SPF cats with FECV strain UCD. Two cats showed mild clinical signs, faecal shedding of infectious virus from 4 dpi, a cell-associated viraemia at inconsistent time points from 5 dpi, a highly neutralising antibody response from 9 dpi, and no major abnormalities in leukocyte numbers. Faecal shedding lasted for 28–56 days, but virus shed during this stage was less infectious in enterocyte cultures and affected by mutations. Remarkably, in the other cat neither clinical signs nor acute shedding were seen, but virus was detected in blood cells from 3 dpi, and shedding of non-enterotropic, mutated viruses suddenly occurred from 14 dpi onwards. Neutralising antibodies arose from 21 dpi. Leukocyte numbers were not different compared to the other cats, except for the CD8+ regulatory T cells. These data indicate that FECV can infect immune cells even in the absence of intestinal replication and raise the hypothesis that the gradual adaptation to these cells can allow non-enterotropic mutants to arise. PMID:26822958

In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

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Oi Kuan Choong

2014-01-01

Full Text Available Feline Infectious Peritonitis (FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV, a virulent mutant strain of Feline Enteric Coronavirus (FECV. Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO RNAs (TFO1 to TFO5, which target the different regions of virulent feline coronavirus (FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

In vitro antiviral activity of circular triple helix forming oligonucleotide RNA towards Feline Infectious Peritonitis virus replication.

Science.gov (United States)

Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman

2014-01-01

Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

Anti-Ma2 antibody related paraneoplastic limbic/brain stem encephalitis associated with breast cancer expressing Ma1, Ma2, and Ma3 mRNAs.

Science.gov (United States)

Sahashi, K; Sakai, K; Mano, K; Hirose, G

2003-09-01

A 69 year old woman presented with cognitive impairment and supranuclear gaze palsy caused by paraneoplastic limbic/brain stem encephalitis associated with atypical medullary breast carcinoma. The cerebrospinal fluid from the patient harboured an anti-neuronal cell antibody against Ma2 antigen, but not against Ma1 or Ma3 antigen. Despite the antibody being restricted to the Ma2 antigen, the patient's cancer tissue expressed Ma1, Ma2, and Ma3 mRNAs. These results, and the expression of Ma2 mRNA in an atypical medullar breast carcinoma in another patient without paraneoplastic encephalitis, indicate that the induction of anti-Ma2 antibody depends on host immunoreponsiveness and not on the presence of the antigen itself in the cancer.

Analysis of transcription factor mRNAs in identified oxytocin and vasopressin magnocellular neurons isolated by laser capture microdissection.

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Madison Humerick

Full Text Available The oxytocin (Oxt and vasopressin (Avp magnocellular neurons (MCNs in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON, and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM, and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun are differentially expressed in the Oxt and Avp MCNs.

Kinetic parameters from thermogravimetric analysis

Science.gov (United States)

Kiefer, Richard L.

1993-01-01

High performance polymeric materials are finding increased use in aerospace applications. Proposed high speed aircraft will require materials to withstand high temperatures in an oxidative atmosphere for long periods of time. It is essential that accurate estimates be made of the performance of these materials at the given conditions of temperature and time. Temperatures of 350 F (177 C) and times of 60,000 to 100,000 hours are anticipated. In order to survey a large number of high performance polymeric materials on a reasonable time scale, some form of accelerated testing must be performed. A knowledge of the rate of a process can be used to predict the lifetime of that process. Thermogravimetric analysis (TGA) has frequently been used to determine kinetic information for degradation reactions in polymeric materials. Flynn and Wall studied a number of methods for using TGA experiments to determine kinetic information in polymer reactions. Kinetic parameters, such as the apparent activation energy and the frequency factor, can be determined in such experiments. Recently, researchers at the McDonnell Douglas Research Laboratory suggested that a graph of the logarithm of the frequency factor against the apparent activation energy can be used to predict long-term thermo-oxidative stability for polymeric materials. Such a graph has been called a kinetic map. In this study, thermogravimetric analyses were performed in air to study the thermo-oxidative degradation of several high performance polymers and to plot their kinetic parameters on a kinetic map.