bluuebunny/crossref_metadata_2025 huggingface dataset:
Symptom induction by Cowpea chlorotic mottle virus on Vigna unguiculata is determined by amino acid residue 151 in the coat protein
Authors: de Assis Filho F. M. , Paguio O. R. , Sherwood J. L. , Deom C. M.
Published: August 2015

The type strain of Cowpea chlorotic mottle virus (CCMV-T) produces a bright chlorosis in cowpea (Vigna unguiculata cv. California Blackeye). The attenuated variant (CCMV-M) induces mild green mottle symptoms that were previously mapped to RNA 3. Restriction fragment exchanges between RNA 3 cDNA clones of CCMV-T and CCMV-M that generate infectious transcripts and site-directed mutagenesis indicated that the codon encoding amino acid residue 151 of the coat protein determines the symptom phenotypes of CCMV-T and CCMV-M. Amino acid 151 is within an α-helical structure required for calcium ion binding and virus particle stability. No differences in virion stability or accumulation were detected between CCMV-T and CCMV-M. Mutational analysis suggested that the amino acid at position 151 and not the nucleotide sequence induce the symptom phenotype. Thus, it is likely that subtle influences by amino acid residue 151 in coat protein–host interactions result in chlorotic and mild green mottle symptoms.
Gill-associated nidovirus of Penaeus monodon prawns transcribes 3′-coterminal subgenomic mRNAs that do not possess 5′-leader sequences
Authors: Cowley Jeff A. , Dimmock Christine M. , Walker Peter J.
Published: August 2015

Sequence analysis of the ∼20 kb 5′-terminal portion of the ssRNA genome of gill-associated virus (GAV) of Penaeus monodon prawns has previously established that it contains an ORF1a–1b replicase gene equivalent to those of the coronavirus and arterivirus members of the order Nidovirales. Sequence analysis of the remaining ∼6·2 kb of the GAV genome downstream of ORF1a–1b to a 3′-poly(A) tail has identified two highly conserved intergenic sequences in which 29/32 nucleotides are conserved. Northern hybridization using probes to the four putative GAV ORFs and either total or poly(A)-selected RNA identified two 3′-coterminal subgenomic (sg) mRNAs of ∼6 kb and ∼5·5 kb. Primer extension and 5′-RACE analyses showed that the sgmRNAs initiate at the same 5′-AC positions in the central region of the two conserved intergenic sequences. Neither method provided any evidence that the GAV sgmRNAs are fused to genomic 5′-leader RNA sequences as is the case with vertebrate coronaviruses and arteriviruses. Intracellular double-stranded (ds)RNAs equivalent in size to the 26·2 kb genomic RNA and two sgRNAs were also identified by RNase/DNase digestion of total RNA from GAV-infected prawn tissue. The identification of only two sgmRNAs that initiate at the same position in conserved intergenic sequences and the absence of 5′-genomic leader sequences fused to these sgmRNAs confirms that GAV has few genes and suggests that it utilizes a transcription mechanism possibly similar to the vertebrate toroviruses but distinct from coronaviruses and arteriviruses.
Administration to mice of a monoclonal antibody that neutralizes the intracellular mature virus form of vaccinia virus limits virus replication efficiently under prophylactic and therapeutic conditions
Authors: Ramírez Juan C. , Tapia Esther , Esteban Mariano
Published: August 2015

The WHO smallpox eradication program was concluded 21 years ago and the non-vaccinated population is now at risk of poxvirus infections, either by contact with monkeypox or through bioterrorism. Since drugs specific against poxvirus infections are limited, neutralizing monoclonal antibodies (mAbs) that are effectivein vivomay be an important tool in controlling poxvirus infections. To this end, we studied the efficacy of the mAb C3, reactive against the trimeric 14-kDa protein of vaccinia virus (VV) localized in the membrane of the intracellular form of mature virus, for its ability to neutralize VV infection in mice. The results show that prophylactic as well as therapeutic administration of mAb C3 can be an effective means of control of VV replication within the host. The interval of antibody efficacy following a single administration, before and after VV inoculation, has been defined. This study reinforces the notion that neutralizing mAbs should be developed to control health-related human infections by poxviruses.
Comparison of neuropathogenicity of poliovirus in two transgenic mouse strains expressing human poliovirus receptor with different distribution patterns
Authors: Ida-Hosonuma Miki , Iwasaki Takuya , Taya Choji , Sato Yuko , Li Jifen , Nagata Noriyo , Yonekawa Hiromichi , Koike Satoshi
Published: August 2015

In order to determine the influence of poliovirus receptor (PVR) expression on poliovirus cell tropism and neuropathogenesis, two transgenic (tg) mouse models were produced in which PVR was expressed under the transcriptional control of the human PVR gene promoter (hg–PVR mice) and the CAG promoter (CAG–PVR mice). Then the pathogenicity of poliovirus after intracerebral inoculation of the type 1 Mahoney strain was compared. These showed completely different clinical and pathological changes. In the former, the expression of PVR in neurons in the central nervous system (CNS) confered susceptibility to poliovirus, and a paralytic disease that resembled the human poliomyelitis occurred. In the latter, PVR expression was detected in glial and ependymal cells in addition to the neurons. Paralysis of the limbs and death were rarely observed and mice survived without showing substantial clinical abnormality. Histopathological examination revealed that glial and ependymal cells also became susceptible to poliovirus infection. Poliovirus antigens were mainly detected in ependymal and glial cells and hippocampal neurons near the lateral ventricles in the brain, but were not frequently detected in neurons in the brainstem unlike in the hg–PVR mice. The levels of viral antigens and virus recovered from the CNS of CAG–PVR mice began to decrease as early as 2 days after inoculation, which suggested induction of a fast immune response. These results suggest that the neuropathogenicity of poliovirus changes markedly depending on the specific expression of the PVR molecule in the CNS.
Characterization of HLA-A2.1-restricted epitopes, conserved in both Hantaan and Sin Nombre viruses, in Hantaan virus-infected patients
Authors: Lee Ki-Young , Chun Eunyoung , Kim Na-Yeon , Seong Baik L.
Published: August 2015

Nine different CTL epitopes, conserved in both Hantaan virus (HTNV) and Sin Nombre virus (SNV), were selected for study. The binding affinity of each peptide with HLA-A2.1 moleculesin vitrowas determined and antigen-specific responses from seven donors who had a previous field infection with HTNV were examined. Although the strength or frequency of CTL activity showed different patterns in the seven patients, five of seven patients showed significant activity against at least one or more epitope peptides. In particular, the peptide ILQDMRNTI (HTNV, aa 334–342; SNV, aa 333–341), which elicited CTL activity in five patients, was shown to be specifically HLA-A2.1-restricted in partially cloned CD8+T cells and also induced activated and effector CD8+T cell-producing T cytotoxic (Tc) type 1 cytokines, such as IL-2 and IFN-γ. The results suggest that this epitope would serve as a useful component for the intervention of both HTNV and SNV infection.
Measles virus-induced modulation of host-cell gene expression
Authors: Bolt Gert , Berg Kurt , Blixenkrone-Møller Merete
Published: August 2015

The influence of measles virus (MV) infection on gene expression by human peripheral blood mononuclear cells (PBMCs) was examined with cDNA microarrays. The mRNA levels of more than 3000 cellular genes were compared between uninfected PBMCs and cells infected with either the Edmonston MV strain or a wild-type MV isolate. The MV-induced upregulation of individual genes identified by microarray analyses was confirmed by RT–PCR. In the present study, a total of 17 genes was found to be upregulated by MV infection. The Edmonston strain grew better in the PBMC cultures than the wild-type MV, and the Edmonston strain was a stronger inducer of the upregulated host cell genes than the wild-type virus. The anti-apoptotic B cell lymphoma 3 (Bcl-3) protein and the transcription factor NF-κB p52 subunit were upregulated in infected PBMCs both at the mRNA and at the protein level. Several genes of the interferon system including that for interferon regulatory factor 7 were upregulated by MV. The genes for a number of chaperones, transcription factors and other proteins of the endoplasmic reticulum stress response were also upregulated. These included the gene for the pro-apoptotic and growth arrest-inducing CHOP/GADD153 protein. Thus, the present study demonstrated the activation by MV of cellular mechanisms and pathways that may play a role in the pathogenesis of measles.
Mutational analysis of a human immunodeficiency virus type 1 Tat protein transduction domain which is required for delivery of an exogenous protein into mammalian cells
Authors: Park Jinseu , Ryu Jiyoon , Kim Kyeong-Ae , Lee Hak Joo , Bahn Jae Hoon , Han Kyuhyung , Choi Eui Yul , Lee Kil Soo , Kwon Hyeok Yil , Choi Soo Young
Published: August 2015

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains a high proportion of arginine and lysine residues, is responsible for highly efficient protein transduction through the plasma membrane. To identify the role of the PTD sequence motif in transduction, various deletions and substitutions were introduced into the PTD. Tat–green fluorescent protein (GFP) fusion proteins, containing various lengths of the Tat PTD, were expressed and the extent of their transduction into mammalian cells was analysed by Western blot analysis and fluorescence microscopy. Deletion analysis of PTD mapped to a nine amino acid motif (residues 49–57: RKKRRQRRR) sufficient for transduction. Further deletion of this Tat basic domain either at the N terminus or at the C terminus significantly decreased transduction efficiency. The transduction efficiencies of GFPs fused to nine consecutive lysine (9Lys–GFP) or arginine (9Arg–GFP) residues were similar to that of Tat(49–57)–GFP. The transduced proteins localized to both the nucleus and the cytosol, as assessed by confocal microscopy and Western blot analysis of subcellular fractions from transduced cells. Thus, the availability of recombinant GFP fusion proteins facilitates the simple and specific identification of protein transduction mediated by these peptide sequences. The modified PTD sequences designed in this study may provide useful tools necessary for delivering therapeutic proteins/peptides into cells.
Initial infection of roots and leaves reveals different resistance phenotypes associated with coat protein gene-mediated resistance to Potato mop-top virus
Authors: Germundsson Anna , Sandgren Maria , Barker Hugh , Savenkov Eugene I. , Valkonen Jari P. T.
Published: August 2015

Resistance to the pomovirus Potato mop-top virus (PMTV) was studied in potato (Solanum tuberosum cv. Saturna) and Nicotiana benthamiana transformed with the coat protein (CP) gene of PMTV. The incidence of PMTV infections was reduced in tubers of the CP-transgenic potatoes grown in the field in soil infested with the viruliferous vector, Spongospora subterranea. However, in those tubers that were infected, all three virus RNAs were detected and virus titres were high. The CP-transgenic N. benthamiana plants were inoculated with PMTV using two methods. Following mechanical inoculation of leaves, no RNA 3 (the CP-encoding RNA homologous to the transgene) was detected in leaves, but in some plants low amounts of RNA 3 were detected in roots; RNA 2 was readily detected in leaves and roots of several plants. Inoculation of roots using viruliferous S. subterranea resulted in infection of roots in all plants and the three PMTV RNAs were detected. However, no systemic movement of PMTV from roots to the above-ground parts was observed, indicating a novel expression of resistance. These data indicate that the CP gene-mediated resistance to PMTV specifically restricts accumulation of PMTV RNA 3, and is more effective in leaves than roots. Furthermore, expression of resistance is different depending on whether leaves or roots are inoculated. Data do not exclude the possibility that both a protein-mediated and an RNA-mediated resistance mechanism are involved.
Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor
Authors: Vanlandschoot Peter , Van Houtte Freya , Roobrouck Annelies , Farhoudi Ali , Leroux-Roels Geert
Published: August 2015

During hepatitis B virus (HBV) infection, high numbers of non-infectious HBV surface antigen (HBsAg) particles are present in circulation. It is shown here that recombinant HBsAg (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. Attachment of rHBsAg to the THP-1 pre-monocytic cell line occurs upon 1,25-dihydroxyvitamin D3-induced differentiation. Binding to monocytes is enhanced by a heat-labile serum protein and is inhibited by Ca2+/Mg2+, low pH and an HBsAg-specific monoclonal antibody. Furthermore, it is shown that rHBsAg suppresses lipopolysaccharide- and IL-2-induced production of cytokines. These results suggest the existence of a monocyte-specific receptor, the engagement of which by HBsAg suppresses the activity of these cells.
Genomic characterization of TT viruses (TTVs) in pigs, cats and dogs and their relatedness with species-specific TTVs in primates and tupaias
Authors: Okamoto Hiroaki , Takahashi Masaharu , Nishizawa Tsutomu , Tawara Akio , Fukai Katsuhiko , Muramatsu Umetaro , Naito Yoshihisa , Yoshikawa Akira
Published: August 2015

Using PCR with primers derived from a non-coding region of the human TT virus (TTV) genome, the TTV sequence in serum samples obtained from pigs (Sus domesticus), dogs (Canis familiaris) and cats (Felis catus) was identified and the entire genomic sequence was determined for each representative isolate. Three TTV isolates (Sd-TTV31 from a pig, Cf-TTV10 from a dog and Fc-TTV4 from a cat) comprising 2878, 2797 and 2064 nucleotides, respectively, each had three open reading frames (ORFs) encoding 436–635 (ORF1), 73–105 (ORF2) and 224–243 (ORF3) aa but lacked ORF4, similar to tupaia TTV. ORF3 was presumed to arise from a splicing of TTV mRNA, similar to human prototype TTV. Although the nucleotide sequence of Sd-TTV31, Cf-TTV10 and Fc-TTV4 differed by more than 50% from each other and from previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates as well as tupaia TTVs of 2·2 kb, they resembled known TTVs and TLMVs with regard to genomic organization and presumed transcriptional profile rather than animal circoviruses of 1·7–2·3 kb. Phylogenetic analysis revealed that Sd-TTV31, Cf-TTV10 and Fc-TTV4 were closer to TTVs from lower-order primates and tupaias than to TTVs from higher-order primates and TLMVs. These results indicate that domestic pigs, cats and dogs are naturally infected with species-specific TTVs with small genomic size and suggest a wide distribution of TTVs with extremely divergent genomic sequence and length in animals.
A model for the generation of multiple A to G transitions in the human respiratory syncytial virus genome: predicted RNA secondary structures as substrates for adenosine deaminases that act on RNA
Authors: Martínez Isidoro , Melero José A.
Published: August 2015

Human respiratory syncytial virus (HRSV) escape mutants selected with antibodies specific for the attachment (G) protein contain diverse genetic alterations, including point mutations, premature stop codons, frame shift changes and A to G hypermutations. The latter changes have only been found in mutants selected with antibodies directed against the conserved central region of the G protein. This gene segment fulfils substrate requirements for adenosine deaminases that act on RNA (ADARs): i.e. it is an A+U rich region of 137 residues and 98 or 106 of them – for A/Mon/3/88 or Long HRSV strains, respectively – are predicted to form intramolecular base pairs leading to a stable RNA secondary structure. In addition, when sequences of the G gene from natural isolates are compared in terms of pairwise substitutions, A to G+G to A changes are preferentially observed in regions where stable intramolecular dsRNA secondary structures are predicted to occur. In this study, a model is proposed in which, in addition to nucleotide misincorporations, reiterative A to G changes in HRSV are generated by ADAR activity operating in short segments (100–200 ribonucleotide residues) of the HRSV genome with high tendency for intramolecular base pairing.
Primary herpes simplex virus type 1 infection of the eye triggers similar immune responses in the cornea and the skin of the eyelids
Authors: Stumpf Thomas H. , Case Rachel , Shimeld Carolyn , Easty David L. , Hill Terry J.
Published: August 2015

Herpetic stromal keratitis (HSK) and blepharoconjunctivitis in humans are thought partly to result from immunopathological responses to herpes simplex virus type 1 (HSV-1). The corneas of NIH mice were inoculated with HSV-1 (strain McKrae) and mice were examined for signs of disease and infection on days 1, 4, 7, 10, 14 and 21. The eyes and eyelids of infected and control mice were processed for immunohistochemistry and double stained for viral antigens and one of the following cell surface markers (Gr-1, F4/80, CD4, CD8, CD45R or MHC class II) or one of the following cytokines (IL-2, IL-4, IL-6, IL-10, IL-12 or IFN-γ). All infected mice developed signs of HSK by day 4 and blepharitis by day 7 and these both persisted until day 21, when signs of resolution where apparent. Virus was detected during the first week of infection and became undetectable by day 10. Large numbers of Gr-1+cells (neutrophils) infiltrated infected corneas and eyelids in areas of viral antigen and CD4+T cells increased significantly in number after virus clearance. In both sites, the predominant cytokines were IL-6, IL-10, IL-12 and IFN-γ, with few IL-2+and IL-4+cells. These observations suggest that the immune responses in the cornea are similar to those in the eyelids but, overall, the responses are not clearly characterized as either Th1 or Th2. In both sites, the neutrophil is the predominant infiltrating cell type and is a likely source of the cytokines observed and a major effector of the disease process.
Characterization of an Epstein–Barr virus-related gammaherpesvirus from common marmoset (Callithrix jacchus)
Authors: Jenson Hal B. , Ench Yasmin , Zhang Yanjin , Gao Shou-Jiang , Arrand John R. , Mackett Michael
Published: August 2015

A gammaherpesvirus related to Epstein–Barr virus (EBV;Human herpesvirus 4) infects otherwise healthy common marmosets (Callithrix jacchus). Long-term culture of common marmoset peripheral blood lymphocytes resulted in outgrowth of spontaneously immortalized lymphoblastoid cell lines, primarily of B cell lineage. Electron microscopy of cells and supernatants showed herpesvirus particles. There were high rates of serological cross-reactivity to other herpesviruses (68–86%), but with very low geometric mean antibody titres [1:12 to human herpesvirus 6 and 1:14 to Herpesvirus papio (Cercopithecine herpesvirus 12)]. Sequence analysis of the conserved herpesvirus DNA polymerase gene showed that the virus is a member of the lymphocryptovirus subgroup and is most closely related to a lymphocryptovirus from rhesus macaques and is closely related to EBV and Herpesvirus papio. High seroprevalence (79%, with geometric mean antibody titre of 1:110) among 28 common marmosets from two geographically distinct colonies indicated that the virus is likely present in many common marmosets in captivity. A New World primate harbouring a lymphocryptovirus suggests that this subgroup arose much earlier than previously thought.
Phylogenetic evidence for adaptive evolution of dengue viruses in nature
Authors: Twiddy S. Susanna , Woelk Christopher H. , Holmes Edward C.
Published: August 2015

A maximum-likelihood approach was used to analyse selection pressures acting on genes from all four serotypes of dengue virus (DEN). A number of amino acid positions were identified within the envelope (E) glycoprotein that have been subject to relatively weak positive selection in both DEN-3 and DEN-4, as well as in two of the five genotypes of DEN-2. No positive selection was detected in DEN-1. In accordance with the function of the E protein as the major antigenic determinant of DEN, the majority of these sites were located in, or near to, potential T- or B-cell epitopes. A smaller number of selected sites was located in other well-defined functional domains of the E protein, suggesting that cell tropism and virus-mediated membrane fusion may also confer fitness advantages to DEN in nature. Several positively selected amino acid substitutions were also identified in the NS2B and NS5 genes of DEN-2, although the cause of this selection is unclear, whereas the capsid, membrane and non-structural genes NS1, NS2A, NS3 and NS4 were all subject to strong functional constraints. Hence, evidence was found for localized adaptive evolution in natural isolates of DEN, revealing that selection pressures differ among serotypes, genotypes and viral proteins.
Cell surface receptors, virus entry and tropism of primate lentiviruses
Authors: Clapham Paul R. , McKnight Áine
Published: August 2015

Human immunodeficiency virus (HIV) exploits cell surface receptors to attach to and gain entry into cells. The HIV envelope spike glycoprotein on the surface of virus particles binds both CD4 and a seven-transmembrane coreceptor. These interactions trigger conformational changes in the envelope spike that induce fusion of viral and cellular membranes and entry of the viral core into the cell cytoplasm. Other cell surface receptors also interact with gp120 and aid attachment of virus particles. This review describes these receptors, their roles in HIV entry and their influence on cell tropism.
An ex vivo murine model to study poliovirus-induced apoptosis in nerve cells
Authors: Couderc Thérèse , Guivel-Benhassine Florence , Calaora Viviane , Gosselin Anne-Sophie , Blondel Bruno
Published: August 2015

Paralytic poliomyelitis results from destruction of motor neurons owing to poliovirus (PV) replication. Using a mouse model, we have previously shown that PV kills neurons of the central nervous system (CNS) as a result of apoptosis (Girard et al., Journal of Virology 73, 6066–6072, 1999). We report the development of mixed mouse primary nerve cell cultures from the cerebral cortex of neonatal mice transgenic for the human PV receptor. These cultures contained all three main cell types of the CNS, i.e. neurons, astrocytes and oligodendrocytes. All three cell types were susceptible to PV infection and virus replication in the cultures led to DNA fragmentation characteristic of apoptosis. PV-induced apoptosis was inhibited by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD.FMK), indicating that this process involved caspases. Thus, these mixed mouse primary nerve cell cultures are a new in vitro model for studying the molecular mechanisms of PV-induced apoptosis in nerve cells.
Generation of a permanent cell line that supports efficient growth of Marek′s disease virus (MDV) by constitutive expression of MDV glycoprotein E
Authors: Schumacher Daniel , Tischer B. Karsten , Teifke Jens-Peter , Wink Kerstin , Osterrieder Nikolaus
Published: August 2015

A recombinant cell line (SOgE) was established, which was derived from the permanent quail muscle cell line QM7 and constitutively expressed the glycoprotein E (gE) gene of Marek′s disease virus serotype 1 (MDV-1). The SOgE cell line supported growth of virulent (RB-1B) and vaccine (CVI988, 584Ap80C) MDV-1 strains at a level comparable with that of primary chicken embryo cells (CEC). The SOgE cell line was used to produce a vaccine against Marek′s disease. Chickens were immunized at 1 day old with 103 p.f.u. CVI988 produced on either CEC or SOgE cells. Challenge infection was performed at day 12 with hypervirulent Italian MDV-1 strain EU1. Whereas 7/7 or 6/6 animals, respectively, immunized with SOgE or QM7 cells alone developed Marek′s disease, only 1/8 animals from both CVI988-immunized groups exhibited signs of disease, suggesting that SOgE cells are a valuable permanent cell culture system for MDV-1 vaccine production.
Molecular characterization of three newly recognized rat parvoviruses
Authors: Wan Cho-Hua , Söderlund-Venermo Maria , Pintel David J. , Riley Lela K.
Published: August 2015

Rodent parvoviruses have been documented to interfere with bothin vivoandin vitroresearch. In this study, three rat parvoviruses distinct from previously characterized rodent parvoviruses were identified from naturally infected rats obtained from four discrete sources. These three newly recognized parvoviruses were designated rat minute virus (RMV)-1a, -1b and -1c. In this study, the genomic nucleotide sequence and the predicted amino acid sequences of proteins for each of the three RMV-1 variants and Kilham rat virus (KRV) were determined and compared with previously characterized rodent parvoviruses. The three RMV-1 variants were shown to be closely related to each other, to be distinct from but closely related to KRV and H-1 virus, and to be significantly different from the previously identified rat parvovirus isolate, RPV-1a.
Circulating and cell-bound antibodies increase coxsackievirus B4-induced production of IFN-α by peripheral blood mononuclear cells from patients with type 1 diabetes
Authors: Hober Didier , Chehadeh Wassim , Weill Jacques , Hober Christine , Vantyghem Marie-Christine , Gronnier Pascale , Wattré Pierre
Published: August 2015

Increased levels of IFN-α have been found in patients with type 1 diabetes who have detectable levels of coxsackievirus B4 (CVB4) RNA in their blood. The IFN-α-inducing activity of CVB4in vitrois weak but can be enhanced by human IgGs. Therefore, it was investigatedin vitrowhether a preferential IFN-α response of peripheral blood mononuclear cells (PBMCs) to CVB4 exists in patients with type 1 diabetes (n=56) compared with healthy subjects (n=20) and whether antibodies play a role. In patients, the levels of IFN-α obtained after stimulation by PBMCs with CVB4 were higher (P=0·008), an individual IFN-α response by PBMCs to CVB4 was more frequent (P=0·0004) and increased levels of IFN-α were observed in CVB4-infected whole blood cultures. The IFN-α-inducing activity of patients plasma and IgGs mixed with CVB4 and then added to PBMCs was high in comparison with healthy subjects (P<0·001) and was inhibited by preincubating the cells with anti-FcγRII, anti-FcγRIII and anti-CAR (coxsackievirus and adenovirus receptor) antibodies. The strong IFN-α responsiveness of PBMCs to CVB4 suggested that IgGs bound to the cell surface might play a role. A short 56 °C incubation of PBMCs from patients responsive to CVB4 generated supernatants, which, when added to cells, exhibited IFN-α-enhancing activity in combination with CVB4, whereas those of controls did not. Specific antibodies for FcγRI, FcγRII and CAR inhibited this activity. These studies demonstrate that CVB4, through interactions with circulating and/or cell-bound IgGs, can strongly induce the production of IFN-α by PBMCs from patients with type 1 diabetes.
Natural genetic recombination between co-circulating heterotypic enteroviruses
Authors: Oprisan Gabriela , Combiescu Mariana , Guillot Sophie , Caro Valerie , Combiescu Andrei , Delpeyroux Francis , Crainic Radu
Published: August 2015

Natural recombination in poliovirus is a frequent phenomenon. In practice, whenever different genotypes have the opportunity to infect the same individual, a high proportion of viruses with recombinant genomes are excreted. To determine whether enteroviruses other than poliovirus can naturally produce viable virions with recombinant genomes, we studied the molecular features of two distant regions of the viral genomes – the VP1 coding region and the 3D polymerase coding region – of the echovirus serotypes associated with a large outbreak of aseptic meningitis. Nucleotide sequences of nine epidemic strains [belonging to echovirus serotypes 4 (E4), 7 (E7) and 30 (E30)] in the two genomic regions (300 nt of VP1 and 520 nt of 3D polymerase) were compared to prototype and field strains, and phylogenetic trees were generated from alignments. In the VP1 region, each of the three epidemic serotypes clustered with the homotypic prototype strain, whereas in the 3D polymerase region, E7 and E30 grouped as a single cluster, distant from the two corresponding prototype strains. This suggests that one of these two E7 and E30 strains has evolved through recombination with the other or that both have acquired the 3D polymerase coding region from a common ancestor. Our results suggest that such genetic recombinations between different echovirus serotypes are possible when multiple epidemic strains are circulating simultaneously.
Identification of R-peptides in envelope proteins of C-type retroviruses
Authors: Bobkova Maria , Stitz Joern , Engelstädter Martin , Cichutek Klaus , Buchholz Christian J.
Published: August 2015

Activation of the murine leukaemia virus (MLV) envelope protein (Env) requires proteolytic cleavage of the R-peptide, a 16 amino acid C-terminal part of the cytoplasmic tail (C-tail) of Env. This paper demonstrates the presence of R-peptides in Env proteins of C-type retroviruses of simian, avian and porcine origin. Sequence alignment with the MLV C-tail led to the identification of a conserved hydrophobic protease cleavage motif located in the centre of retroviral Env protein C-tails. Expression of Env proteins, truncated at the predicted cleavage sites, of spleen necrosis virus (SNV), gibbon ape leukaemia virus and porcine endogenous retroviruses resulted in cell–cell fusion as monitored by microscopy and reporter gene fusion assays. Western blot analysis of MLV particles pseudotyped with the SNV Env protein demonstrated proteolytic cleavage of the SNV R-peptide by the MLV protease. Our data suggest that activation of membrane fusion by R-peptide cleavage is a common mode in C-type retroviruses.
Analysis of mouse polyomavirus mutants with lesions in the minor capsid proteins
Authors: Mannová Petra , Liebl David , Krauzewicz Nina , Fejtová Anna , Štokrová Jitka , Palková Zdena , Griffin Beverly E. , Forstová Jitka
Published: August 2015

Polyomavirus mutants E, Q and H, expressing non-myristylated VP2, were generated by replacing the N-terminal glycine residue with glutamic acid, glutamine or histidine, respectively. Viruses mutated in either VP2 or VP3 translation initiation codons were also prepared. All mutated genomes, when transfected into murine host cells, gave rise to viral particles. Infectivity of VP2−and VP3−viruses, as measured by the number of cells expressing viral antigens, was dramatically diminished, indicative of defects in the early stages of infection. In contrast, the absence of a myristyl moiety on VP2 did not substantially affect the early steps of virus infection. No differences in numbers of cells expressing early or late viral antigens were observed between wild-type (wt) and E or Q myr−viruses during the course of a life cycle. Furthermore, no delay in virus DNA replication was detected. However, when cells were left for longer in culture, the number of infected cells, measured by typical virus bursts, was much lower when mutant rather than wt genomes were used.In situ, cell fractionation studies revealed differences in the interaction of viral particles with host cell structures. The infectivity of mutants was affected not only by loss of the myristyl group on VP2, but also, and to a greater extent, by alterations of the N-terminal amino acid composition.
Kinetics of binding, uptake and degradation of live fluorescent (DsRed) bacteria by Dictyostelium discoideum
Authors: Maselli Andrew , Laevsky Gary , Knecht David A
Published: August 2015

The kinetics of binding, uptake and degradation of bacteria by vegetativeDictyosteliumamoeba usingEscherichia coliexpressing the recombinant fluorescent protein DsRed have been characterized. There are significant advantages to using DsRed-expressing bacteria for phagocytosis assays. Stable expression of the fluorescent protein, DsRed, provides living bacteria with a bright internal fluorescent signal that is degradable in the phagolysosomal pathway. Unlike assays with chemically labelled bacteria or latex beads, the bacteria are alive and possess a natural, unaltered external surface for receptor interaction.Dictyosteliumcells rapidly bind and phagocytose DsRed bacteria. Pulse–chase experiments show that the signal derived from DsRed is degraded with a half-life of approximately 45 min. To distinguish internalized bacteria from those bound to the surface, an assay was developed in which sodium azide was used to release surface-bound particles. Surprisingly, surface particle release appears to be independent of myosin II function. Using this assay it was shown that the uptake of bacteria into cells is extremely rapid. After 1 min incubation, 20% of the signal is derived from internalized bacteria. The proportion of the signal from internalized bacteria increases gradually and reaches 50% at steady state. This assay will be useful in investigations of the molecular machinery of phagocytosis and post-internalization vesicle trafficking.
rDNA analyses of planktonic heterocystous cyanobacteria, including members of the genera Anabaenopsis and Cyanospira The GenBank accession numbers of the 16S rDNA gene sequences reported in this paper are AY038032–AY038037.
Authors: Iteman Isabelle , Rippka Rosmarie , Tandeau de Marsac Nicole , Herdman Michael
Published: August 2015

The taxonomic coherence and phylogenetic relationships of 11 planktonic heterocystous cyanobacterial isolates were examined by investigating two areas of the rRNA operon, the 16S rRNA gene (rrnS) and the internal transcribed spacer (ITS) located between the 16S rRNA and 23S rRNA genes. TherrnSsequences were determined for five strains, including representatives ofAnabaenaflos-aquae,Aphanizomenonflos-aquae,Nodulariasp. and two alkaliphilic planktonic members of the generaAnabaenopsisandCyanospira, whose phylogenetic position was previously unknown. Comparison of the data with those previously published for individual groups of planktonic heterocystous cyanobacteria showed that, with the exception of members assigned to the genusCylindrospermopsis, all the planktonic strains form a distinct subclade within the monophyletic clade of heterocystous cyanobacteria. Within this subclade five different phylogenetic clusters were distinguished. The phylogenetic groupings ofAnabaenaandAphanizomenonstrains within three of these clusters were not always consistent with their generic or specific assignments based on classical morphological definitions, and the high degree of sequence similarity between strains ofAnabaenopsisandCyanospirasuggests that they may be assignable to a single genus. Ribotyping and additional studies performed on PCR amplicons of the 16S rDNA or the ITS for the 11 planktonic heterocystous strains demonstrated that they all contain multiplerrnoperons and ITS regions of variable size. Finally, evidence is provided for intra-genomic sequence heterogeneity of the 16S rRNA genes within most of the individual isolates.
Signal-sequence-independent secretion of the staphylococcal nuclease in Mycobacterium smegmatis
Authors: Recchi Chiara , Rauzier Jean , Gicquel Brigitte , Reyrat Jean-Marc
Published: August 2015

Staphylococcus aureus nuclease is a small, secreted protein which has been successfully used as a reporter system to identify exported products in Lactococcus lactis. Here, biochemical evidence is provided that the nuclease is exported by Mycobacterium smegmatis in the presence, but also in the absence of a signal sequence, and thus probably independently of the Sec translocation pathway. This implies that the nuclease should not be used as a reporter system in mycobacteria for the identification of exported products, despite what has been reported previously in the literature. The nuclease can be extended to create hybrid proteins that remain compatible with its secretion, whereas some other shorter fusions are not tolerated. This suggests that correct folding is required for efficient export. Extensive mutational analysis did not identify a specific secretion pathway. This suggests that the nuclease may be exported by different redundant systems or that components of this alternative Sec pathway are essential for bacterial survival.
Pseudoclavibacter terrae sp. nov. isolated from rhizosphere soil of Ophiopogon japonicus
Authors: Du Juan , Singh Hina , Yang Jung-Eun , Shik Yin Chang , Kook MooChang , Yu Hongshan , Yi Tae-Hoo
Published: August 2015

Strain THG-MD12T, a Gram-reaction-positive, aerobic, non-motile, rod-shaped bacterium was isolated from rhizosphere soil of Ophiopogon japonicus in PR China. THG-MD12T was closely related to members of the genus Pseudoclavibacter and showed the highest 16S rRNA gene sequence similarities with Pseudoclavibacter helvolus KCTC 19531T (98.8 %) and Pseudoclavibacter chungangensis KCTC 22691T (96.9 %). DNA–DNA hybridization showed 41.9 ± 2.1 % and 12.4 ± 0.9 % DNA reassociation with P. helvolus KCTC 19531T and P. chungangensis KCTC 22691T, respectively. Chemotaxonomic analyses revealed that strain THG-MD12T possesses menaquinone-9 as the predominant respiratory quinone, 2,4-diaminobutyric acid as the diamino acid in the peptidoglycan and anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and anteiso-C17 : 0 as the major fatty acids. The polar lipid profile was found to consist of diphosphatidylglycerol, phosphatidylglycerol, two unknown glycolipids and two unknown lipids. These data corroborated the affiliation of THG-MD12T to the genus Pseudoclavibacter. Thus, the isolate represents a novel species, for which the name Pseudoclavibacter terrae sp. nov. is proposed, with THG-MD12T as the type strain ( = CCTCC AB 2015124T = KCTC 39562T).
Confluentimicrobium naphthalenivorans sp. nov., a naphthalene-degrading bacterium isolated from sea-tidal-flat sediment, and emended description of the genus Confluentimicrobium Park et al. 2015
Authors: Jeong Hye Im , Jin Hyun Mi , Jeon Che Ok
Published: August 2015

A Gram-staining-negative, aerobic and moderately halophilic bacterium, designated strain NS6T, was isolated from sea-tidal-flat sediment of the South Sea in Korea. Cells were motile rods with a single flagellum showing positive catalase and oxidase activities. Strain NS6Tgrew at 15–40 °C (optimum, 30 °C), at pH 6.0–9.0 (optimum, pH 7.0–7.5) and in the presence of 1–5 % (w/v) NaCl (optimum, 2 %). Ubiquinone-10 was identified as the only isoprenoid quinone and summed feature 8 (comprising C18 : 1ω7c/ω6c), C19 : 0cyclo ω8c, C16 : 0and C10 : 03-OH were the major fatty acids. Strain NS6Tcontained phosphatidylglycerol, sulfoquinovosyldiacylglycerol, an unidentified amino lipid and an unidentified lipid as the major polar lipids. An unidentified phospholipid and three unidentified lipids were also detected as the minor polar lipids. The G+C content of the genomic DNA was 64.5 mol%. Strain NS6Twas most closely related toConfluentimicrobium lipolyticumSSK1-4Twith a 96.8 % 16S rRNA gene sequence similarity, and the phylogenetic analysis based on 16S rRNA gene sequences showed that strain NS6Tformed a tight phylogenetic lineage withC. lipolyticumSSK1-4T. The physiological, chemotaxonomic and phylogenetic properties suggest that strain NS6Trepresents a novel species of the genusConfluentimicrobium, for which the nameConfluentimicrobium naphthalenivoranssp. nov. is proposed. The type strain is NS6T( = KCTC 18418T = JCM 30828T). An emended description of the genusConfluentimicrobiumis also proposed.
Alteromonas naphthalenivorans sp. nov., a polycyclic aromatic hydrocarbon-degrading bacterium isolated from tidal-flat sediment
Authors: Mi Jin Hyun , Hyun Kim Kyung , Ok Jeon Che
Published: August 2015

A Gram-staining-negative and halotolerant bacterium, designated SN2T, capable of biodegrading polycyclic aromatic hydrocarbons, was isolated from a tidal flat contaminated with crude oil in Korea. Cells were strictly aerobic, catalase- and oxidase-positive, motile rods, with a single polar flagellum. Growth was observed at 4–37 °C (optimum, 25–30 °C) at pH 6.0–9.0 (optimum, pH 7.0–7.5) and in the presence of 0.5–9.0 % (w/v) NaCl (optimum, 2.0 %). Only ubiquinone 8 was detected as the isoprenoid quinone, and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH), C16 : 0, C18 : 1ω7c and C12 : 0 were observed as the major cellular fatty acids. The major polar lipids consisted of phosphatidylethanolamine, phosphatidylglycerol, a glycolipid, an aminolipid and three unidentified lipids. The DNA G+C content was 43.5 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain SN2T formed a phylogenetic lineage with Alteromonas stellipolaris and Alteromonas addita within the genus Alteromonas, which was consistent with multilocus phylogenetic and MALDI-TOF MS analyses. Strain SN2T was most closely related to the type strains of A. stellipolaris, A. addita and Alteromonas macleodii, with 16S rRNA gene sequence similarities of 99.5, 99.3 and 98.4 % and DNA–DNA relatedness of 48.7 ± 6.6, 24.9 ± 7.5 and 27.9 ± 8.4 %, respectively. In conclusion, strain SN2T represents a novel species of the genus Alteromonas, for which the name Alteromonas naphthalenivorans sp. nov. is proposed. The type strain is SN2T ( = KCTC 11700BPT = JCM 17741T = KACC 18427T).
R2d2 drives selfish sweeps in the house mouse
Authors: Didion John P , Morgan Andrew Parker , Yadgary Liran , Bell Timothy A , McMullan Rachel Clara , Ortiz de Solorzano Lydia , Britton-Davidian Janice , Bult Carol J , Campbell Karl J , Castiglia Riccardo , Ching Yung-Hao , Chunco Amanda J , Crowley James J , Chesler Elissa J , French John E , Gabriel Sofia I , Gatti Daniel M , Garland Theodore , Giagia-Athanasopoulou Eva B , Giménez Mabel D , Grize Sofia A , Gündüz İslam , Holmes Andrew , Hauffe Heidi C , Herman Jeremy S , Holt James M , Hua Kunji , Jolley Wesley J , Lindholm Anna K , López-Fuster María J , Mitsainas George , Mathias Maria , McMillan Leonard , Ramalhinho M Graça , Rehermann Barbara , Rosshart Stephan P , Searle Jeremy B , Shiao Meng-Shin , Solano Emanuela , Svenson Karen L , Thomas-Laemont Pat , Threadgill David W , Ventura Queija Jacint , Weinstock George M , Pomp Daniel , Churchill Gary A , Pardo-Manuel de Villena Fernando
Published: August 2015

A selective sweep is the result of strong positive selection rapidly driving newly occurring or standing genetic variants to fixation, and can dramatically alter the pattern and distribution of allelic diversity in a population or species. Population-level sequencing data have enabled discoveries of selective sweeps associated with genes involved in recent adaptations in many species. In contrast, much debate but little empirical evidence addresses whether "selfish" genes are capable of fixation -- thereby leaving signatures identical to classical selective sweeps -- despite being neutral or deleterious to organismal fitness. We previously reported the discovery of R2d2, a large copy-number variant that causes non-random segregation of mouse Chromosome 2 in females due to meiotic drive. Here we show population-genetic data consistent with a "selfish" sweep driven by alleles of R2d2 with high copy number (R2d2HC) in natural populations of mice. We replicate this finding in multiple closed breeding populations from six outbred backgrounds segregating for R2d2 alleles. We find that R2d2HC rapidly increases in frequency, and in most cases becomes fixed in significantly fewer generations than can be explained by genetic drift. R2d2HC is also associated with significantly reduced litter sizes in heterozygosity, making it a true selfish allele. Our data provide direct evidence of populations actively undergoing selfish sweeps, and demonstrate that meiotic drive can rapidly alter the genomic landscape in favor of mutations with neutral or even negative effects on overall Darwinian fitness. Further study will reveal the incidence of selfish sweeps, and will elucidate the relative contributions of selfish genes, adaptation and genetic drift to evolution.
A novel recombinant DNA system for high efficiency affinity purification of proteins inSaccharomyces cerevisiae
Authors: Carrick Brian H. , Hao Lixuan , Smaldino Philip J. , Engelke David R.
Published: August 2015

Abstract

Isolation of endogenous proteins fromSaccharomyces cerevisiaehas been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. The tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini ofS. cerevisiaeORFs using selection for anat1marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background in a single step. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent will allow isolation of rare or unstable protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or chromatin-associated assemblies.