nonstructural proteins nsp3 and nsp4 of ross river and o'nyong-nyong viruses: sequence and comparison with those of other alphaviruses.we have sequenced the nsp3 and nsp4 region of two alphaviruses, ross river virus and o'nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsp3 and nsp4 in many alphaviruses. we found that ross river virus possesses an in-phase opal termination codon between nsp3 and nsp4, whereas in o'nyong-nyong virus this termination codon is replaced by an arginine codon. previous studies have shown that two other alphaviruses, sindbis ...19882834873
replication of arboviruses in mouse organ cultures. i. results with middelburg virus in organ cultures and peritoneal macrophages. 19744370336
number of plaque forming units of middelburg virus injected by aedes aegypti during feeding. 19704394996
[isolation of a strain of middelburg virus from a batch of aedes (a.) cumminsi gathered in bandia (senegal)]. 19695395495
growth and release of several alphaviruses in chick and bhk cells.the growth and release of several alphaviruses, including several strains of sindbis virus (the wild-type strain, the large plaque and small plaque variants of the hr strain, and the hr mutant ts103), semliki forest virus(sfv) and middelburg virus, and of the unrelated rhabdovirus, vesicular stomatitis virus (vsv), have been compared in chick cells and in bhk-21 cells as a function of the culture conditions for the host cell and the ionic strength of the medium. the small plaque strain of sindbi ...19806255069
sequence coding for the alphavirus nonstructural proteins is interrupted by an opal termination codon.we have obtained the nucleotide sequence of the genomic rnas of two alphaviruses, sindbis virus and middelburg virus, over an extensive region encoding the nonstructural (replicase) proteins. in both viruses in an equivalent position an opal (uga) termination codon punctuates a long otherwise open reading frame. the nonstructural proteins are translated as polyprotein precursors that are processed by posttranslational cleavage into four polypeptide chains; the sequence data presented here indica ...19836577423
sequence-specific recognition of a subgenomic rna promoter by a viral rna polymerase.rna templates of 33 nucleotides containing the brome mosaic virus (bmv) core subgenomic promoter were used to determine the promoter elements recognized by the bmv rna-dependent rna polymerase (rdrp) to initiate rna synthesis. nucleotides at positions -17, -14, -13, and -11 relative to the subgenomic initiation site must be maintained for interaction with the rdrp. changes to every other nucleotide at these four positions allow predictions for the base-specific functional groups required for rdr ...19979326593
functions of conserved motifs in the rna-dependent rna polymerase of a yeast double-stranded rna least eight conserved motifs are visible in the totivirus rna-dependent rna polymerase (rdrp). we have systematically altered each of these in the saccharomyces cerevisiae double-stranded rna virus scvl1 by substituting the conserved motifs from a giardiavirus. the results help define the conserved regions of the rdrp involved in polymerase function and those essential for other reasons.19989557735
replicase complex genes of semliki forest virus confer lethal neurovirulence.semliki forest virus (sfv) is a mosquito-transmitted pathogen of small rodents, and infection of adult mice with sfv4, a neurovirulent strain of sfv, leads to lethal encephalitis in a few days, whereas mice infected with the avirulent a7(74) strain remain asymptomatic. in adult neurons, a7(74) is unable to form virions and hence does not reach a critical threshold of neuronal damage. to elucidate the molecular mechanisms of neurovirulence, we have cloned and sequenced the entire 11,758-nucleotid ...200010775594
evolutionary relationships and systematics of the alphaviruses.partial e1 envelope glycoprotein gene sequences and complete structural polyprotein sequences were used to compare divergence and construct phylogenetic trees for the genus alphavirus. tree topologies indicated that the mosquito-borne alphaviruses could have arisen in either the old or the new world, with at least two transoceanic introductions to account for their current distribution. the time frame for alphavirus diversification could not be estimated because maximum-likelihood analyses indic ...200111581380
alphavirus minus-strand rna synthesis: identification of a role for arg183 of the nsp4 polymerase.a partially conserved region spanning amino acids 142 to 191 of the sindbis virus (sin) nsp4 core polymerase is implicated in host restriction, elongation, and promoter recognition. we extended the analysis of this region by substituting ser, ala, or lys for a highly conserved arg183 residue immediately preceding its absolutely conserved ser184-ala-val-pro-ser188 sequence. in chicken cells, the nsp4 arg183 mutants had a nonconditionally lethal, temperature-sensitive (ts) growth phenotype caused ...200212163582
molecular genetic evidence that the hydrophobic anchors of glycoproteins e2 and e1 interact during assembly of alphaviruses.chimeric alphaviruses in which the 6k and glycoprotein e1 moieties of sindbis virus are replaced with those of ross river virus grow very poorly, but upon passage, adapted variants arise that grow >100 times better. we have sequenced the entire domain encoding the e2, 6k, and e1 proteins of a number of these adapted variants and found that most acquired two amino acid changes, which had cumulative effects. in three independent passage series, amino acid 380 of e2, which is in the transmembrane d ...200212239293
middelburg virus; a hitherto unknown agent isolated from aedes mosquitoes during an epizootic in sheep in the eastern cape province. 195713529199
a conserved histidine in the ij loop of the semliki forest virus e1 protein plays an important role in membrane fusion.the enveloped alphavirus semliki forest virus (sfv) infects cells via a low ph-triggered membrane fusion reaction mediated by the e1 protein. e1 is a class ii fusion protein that contains the hydrophobic fusion peptide loop and converts to a stable homotrimer during the fusion reaction. intriguingly, the fusion loop is closely associated with a loop connecting the i and j beta-strands. this ij loop plays a role in the cholesterol dependence of membrane fusion and is specifically susceptible to p ...200415564465
a family of mammalian e3 ubiquitin ligases that contain the ubr box motif and recognize n-degrons.a subset of proteins targeted by the n-end rule pathway bear degradation signals called n-degrons, whose determinants include destabilizing n-terminal residues. our previous work identified mouse ubr1 and ubr2 as e3 ubiquitin ligases that recognize n-degrons. such e3s are called n-recognins. we report here that while double-mutant ubr1(-/-) ubr2(-/-) mice die as early embryos, the rescued ubr1(-/-) ubr2(-/-) fibroblasts still retain the n-end rule pathway, albeit of lower activity than that of w ...200516055722
phylogenetic analysis of buggy creek virus: evidence for multiple clades in the western great plains, united states of america.we present the first detailed phylogenetic analysis of buggy creek virus (bcrv), a poorly known alphavirus with transmission cycles involving a cimicid swallow bug (oeciacus vicarius) vector and cliff swallows (petrochelidon pyrrhonota) and house sparrows (passer domesticus) as the principal avian hosts. nucleotide sequences of a 2,075-bp viral envelope glycoprotein-coding region, covering the entire pe2 gene, were determined for 33 bcrv isolates taken from swallow bugs at cliff swallow colonies ...200616936062
functions of the stem region of the semliki forest virus fusion protein during virus fusion and assembly.membrane fusion of the alphaviruses is mediated by the e1 protein, a class ii virus membrane fusion protein. during fusion, e1 dissociates from its heterodimer interaction with the e2 protein and forms a target membrane-inserted e1 homotrimer. the structure of the homotrimer is that of a trimeric hairpin in which e1 domain iii and the stem region fold back toward the target membrane-inserted fusion peptide loop. the e1 stem region has a strictly conserved length and several highly conserved resi ...200616971447
complete nucleotide sequence of middelburg virus, isolated from the spleen of a horse with severe clinical disease in zimbabwe.the complete nucleotide sequence of middelburg virus (midv) was determined for strain midv-857 from zimbabwe. the isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that midv can cause severe disease in equids. full-length cdna copies of the viral genome were successfully synthesized by an innovative rt-pcr amplification approach using an 'anchor primer' combined with the smart methodology described previously for the synthesis of ...200717947533
a phylogenomic study of the genus alphavirus employing whole genome comparison.the phylogenetics of the genus alphavirus have historically been characterized using partial gene, single gene or partial proteomic data. we have mined cdna and amino acid sequences from genbank for all fully sequenced and some partially sequenced alphaviruses and generated phylogenomic analyses of the genus alphavirus genus, employing capsid encoding structural regions, non-structural coding regions and complete viral genomes. our studies support the presence of the previously reported recombin ...200518629194
discovery of frameshifting in alphavirus 6k resolves a 20-year enigma.the genus alphavirus includes several potentially lethal human viruses. additionally, species such as sindbis virus and semliki forest virus are important vectors for gene therapy, vaccination and cancer research, and important models for virion assembly and structural analyses. the genome encodes nine known proteins, including the small '6k' protein. 6k appears to be involved in envelope protein processing, membrane permeabilization, virion assembly and virus budding. in protein gels, 6k migrat ...200818822126
the nsp3 macro domain is important for sindbis virus replication in neurons and neurovirulence in mice.sindbis virus (sinv), the prototype alphavirus, contains a macro domain in the highly conserved n-terminal region of nonstructural protein 3 (nsp3). however, the biological role of the macro domain is unclear. mutations of amino acids 10 and 24 from asparagine to alanine in the adp-ribose binding region of the macro domain impaired sinv replication and viral rna synthesis particularly in neurons, but did not alter binding of poly(adp-ribose). mutation at position 10 had the greatest effect and c ...200919395054
replication cycle of chikungunya: a re-emerging arbovirus.arboviruses (or arthropod-borne viruses), represent a threat for the new century. the 2005-2006 year unprecedented epidemics of chikungunya virus (chikv) in the french reunion island in the indian ocean, followed by several outbreaks in other parts of the world such as india, have attracted the attention of clinicians, scientists, and state authorities about the risks linked to this re-emerging mosquito-borne virus. chikv, which belongs to the alphaviruses genus, was not previously regarded as a ...200919732931
fatal human case of western equine encephalitis, the editor: the genus alphavirus (family togaviridae) comprises 29 viral species (1), grouped in at least 7 antigenic complexes by their serologic cross-reactivity (2). they are maintained in nature through enzootic cycles involving arthropods as vectors with subsequent amplification in small mammals or birds, and epizootic cycles between mosquitoes and large mammals such as horses or humans.201121529429
ribosomal frameshifting and transcriptional slippage: from genetic steganography and cryptography to adventitious use.genetic decoding is not 'frozen' as was earlier thought, but dynamic. one facet of this is frameshifting that often results in synthesis of a c-terminal region encoded by a new frame. ribosomal frameshifting is utilized for the synthesis of additional products, for regulatory purposes and for translational 'correction' of problem or 'savior' indels. utilization for synthesis of additional products occurs prominently in the decoding of mobile chromosomal element and viral genomes. one class of re ...201627436286
molecular detection of flaviviruses and alphaviruses in mosquitoes (diptera: culicidae) from coastal ecosystems in the colombian caribbean.arboviruses belonging to the genera flavivirus and alphavirus were detected in mosquitoes in a rural area of san bernardo del viento (córdoba, colombia). a total of 22,180 mosquitoes were collected, sorted into 2,102 pools, and tested by generic/nested reverse transcription-polymerase chain reaction. venezuelan equine encephalitis virus, dengue virus, west nile virus, st. louis encephalitis virus, yellow fever virus, and culex flavivirus were detected and identified by sequencing. the detection ...201627706377
a field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the chikungunya virus.chikungunya virus (chikv) is a mosquito-borne virus currently transmitted in about 60 countries. chikv causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. detection of the infection is mostly done using rt-rcr or elisa, which are not suitable for point-of-care diagnosis.201627685649
sindbis and middelburg old world alphaviruses associated with neurologic disease in horses, south africa.old world alphaviruses were identified in 52 of 623 horses with febrile or neurologic disease in south africa. five of 8 sindbis virus infections were mild; 2 of 3 fatal cases involved co-infections. of 44 middelburg virus infections, 28 caused neurologic disease; 12 were fatal. middelburg virus likely has zoonotic potential.026583836
broadly neutralizing alphavirus antibodies bind an epitope on e2 and inhibit entry and egress.we screened a panel of mouse and human monoclonal antibodies (mabs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. passive transfer of broadly neutralizing mabs protected mice against infection by chikungunya, mayaro, and o'nyong'nyong alphaviruses. using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing mabs block multiple steps in the ...201526553503
evolutionary genetics and vector adaptation of recombinant viruses of the western equine encephalitis antigenic complex provides new insights into alphavirus diversity and host switching.western equine encephalitis virus (weev), highlands j virus (hjv), and fort morgan virus (fmv) are the sole representatives of the wee antigenic complex of the genus alphavirus, family togaviridae, that are endemic to north america. all three viruses have their ancestry in a recombination event involving eastern equine encephalitis virus (eeev) and a sindbis (sin)-like virus that gave rise to a chimeric alphavirus that subsequently diversified into the present-day weev, hjv, and fmv. here, we pr ...201425463613
in- silico exploration of thirty alphavirus genomes for analysis of the simple sequence repeats.the compilation of simple sequence repeats (ssrs) in viruses and its analysis with reference to incidence, distribution and variation would be instrumental in understanding the functional and evolutionary aspects of repeat sequences. present study encompasses the analysis of ssrs across 30 species of alphaviruses. the full length genome sequences, assessed from ncbi were used for extraction and analysis of repeat sequences using imex software. the repeats of different motif sizes (mono- to penta ...201425606453
a genome-wide analysis of rna pseudoknots that stimulate efficient -1 ribosomal frameshifting or readthrough in animal viruses.programmed -1 ribosomal frameshifting (prf) and stop codon readthrough are two translational recoding mechanisms utilized by some rna viruses to express their structural and enzymatic proteins at a defined ratio. efficient recoding usually requires an rna pseudoknot located several nucleotides downstream from the recoding site. to assess the strategic importance of the recoding pseudoknots, we have carried out a large scale genome-wide analysis in which we used an in-house developed program to d ...201324298557
a tyrosine-to-histidine switch at position 18 of the ross river virus e2 glycoprotein is a determinant of virus fitness in disparate hosts.arthritogenic alphaviruses are human pathogens maintained in nature through alternating replication in vertebrates and mosquitoes. using chimeric viruses, we previously reported that replacement of the pe2 coding region of the t48 strain of ross river virus (rrv-t48) with that from the attenuated dc5692 strain, which differ by 7 amino acids, resulted in an attenuated disease phenotype in a mouse model of rrv-induced rheumatic disease. here, we demonstrate that introduction of one of these amino ...201323514884
adaptive changes in alphavirus mrna translation allowed colonization of vertebrate hosts.members of the alphavirus genus are arboviruses that alternate replication in mosquitoes and vertebrate hosts. in vertebrate cells, the alphavirus resists the activation of antiviral rna-activated protein kinase (pkr) by the presence of a prominent rna structure (downstream loop [dlp]) located in viral 26s transcripts, which allows an eif2-independent translation initiation of these mrnas. this article shows that dlp structure is essential for replication of sindbis virus (sinv) in vertebrate ce ...201222761388
cell-based analysis of chikungunya virus e1 protein in membrane fusion.chikungunya fever is a pandemic disease caused by the mosquito-borne chikungunya virus (chikv). e1 glycoprotein mediation of viral membrane fusion during chikv infection is a crucial step in the release of viral genome into the host cytoplasm for replication. how the e1 structure determines membrane fusion and whether other chikv structural proteins participate in e1 fusion activity remain largely unexplored.201222520648
improved assembly procedure of viral rna genomes amplified with phi29 polymerase from new generation sequencing sequencing technologies have opened the way to the discovery and the characterization of pathogenic viruses in clinical samples. however, the use of these new methods can require an amplification of viral rna prior to the sequencing. among all the available methods, the procedure based on the use of phi29 polymerase produces a huge amount of amplified dna. however, its major disadvantage is to generate a large number of chimeric sequences which can affect the assembly step. the pre-process m ...201627605096
an updated checklist of mosquito species (diptera: culicidae) from updated checklist of 235 mosquito species from madagascar is presented. the number of species has increased considerably compared to previous checklists, particularly the last published in 2003 (178 species). this annotated checklist provides concise information on endemism, taxonomic position, developmental stages, larval habitats, distribution, behavior, and vector-borne diseases potentially transmitted. the 235 species belong to 14 genera: aedeomyia (3 species), aedes (35 species), anophel ...201627101839
an rna trapping mechanism in alphavirus mrna promotes ribosome stalling and translation initiation.during translation initiation, eukaryotic initiation factor 2 (eif2) delivers the met-trna to the 40s ribosomal subunit to locate the initiation codon (augi) of mrna during the scanning process. stress-induced eif2 phosphorylation leads to a general blockade of translation initiation and represents a key antiviral pathway in mammals. however, some viral mrnas can initiate translation in the presence of phosphorylated eif2 via stable rna stem-loop structures (dlp; downstream loop) located in thei ...201626984530
complete genome sequences of two middelburg viruses isolated from arthropods in the central african republic.arboviral diseases are a major threat to human and animal health today. analysis of whole-genome sequences of decades-old arboviral strains may bring new insights into the viral evolution that might have facilitated outbreaks. here, we report the whole-genome sequences of two middelburg viruses isolated several decades ago in the central african republic.201425342688
isolates of liao ning virus from wild-caught mosquitoes in the xinjiang province of china in 2005.liao ning virus (lnv) is related to banna virus, a known human-pathogen present in south-east asia. both viruses belong to the genus seadornavirus, family reoviridae. lnv causes lethal haemorrhage in experimentally infected mice. twenty seven isolates of lnv were made from mosquitoes collected in different locations within the xinjiang province of north-western china during 2005. these mosquitoes were caught in the accommodation of human patients with febrile manifestations, or in animal barns w ...201222649554
rtips: fast and accurate tools for rna 2d structure prediction using integer programming.we present a web-based tool set rtips for fast and accurate prediction of rna 2d complex structures. rtips comprises two computational tools based on integer programming, ipknot for predicting rna secondary structures with pseudoknots and ractip for predicting rna-rna interactions with kissing hairpins. both servers can run much faster than existing services with the same purpose on large data sets as well as being at least comparable in prediction accuracy. the rtips web server along with the s ...201222600734
shuni virus as cause of neurologic disease in determine which agents cause neurologic disease in horses, we conducted reverse transcription pcr on isolates from of a horse with encephalitis and 111 other horses with acute disease. shuni virus was found in 7 horses, 5 of which had neurologic signs. testing for lesser known viruses should be considered for horses with unexplained illness.022305525
gene analogue finder: a grid solution for finding functionally analogous gene date more than 2,1 million gene products from more than 100000 different species have been described specifying their function, the processes they are involved in and their cellular localization using a very well defined and structured vocabulary, the gene ontology (go). such vast, well defined knowledge opens the possibility of compare gene products at the level of functionality, finding gene products which have a similar function or are involved in similar biological processes without relyi ...200717767718
chikungunya virus capsid protein contains nuclear import and export signals.chikungunya virus (chikv) is an alphavirus of the togaviridae family. after autoproteolytic cleavage, the chikv capsid protein (cp) is involved in rna binding and assembly of the viral particle. the monomeric cp is approximately 30 kda in size and is small enough for passive transport through nuclear pores. some alphaviruses are found to harbor nuclear localization signals (nls) and transport of these proteins between cellular compartments was shown to be energy dependent. the active nuclear imp ...201323984714
distribution of mosquitoes and mosquito-borne arboviruses in yunnan province near the china-myanmar-laos border.abstract. economic development and increased tourism in the southern region of yunnan province in china, adjacent to several countries in southeast asia, has increased the likelihood of import and export of vectors and vector-borne diseases. we report the results of surveillance of mosquitoes and mosquito-borne arboviruses along the border of china-myanmar-laos in 2005 and 2006, and information associating several arboviruses with infections and possibly disease in local human populations. seven ...201121540383
e1 mutants identify a critical region in the trimer interface of the semliki forest virus fusion protein.the alphavirus semliki forest virus (sfv) uses a membrane fusion reaction to infect host cells. fusion of the virus and cell membranes is triggered by low ph in the endosome and is mediated by the viral membrane protein e1. during fusion, e1 inserts into the target membrane, trimerizes, and refolds into a hairpin conformation. formation of the e1 homotrimer is critical to membrane fusion, but the mechanism of trimerization is not understood. the crystal structure of the postfusion e1 trimer show ...200919692469
functional analysis of nsp3 phosphoprotein mutants of sindbis virus.alphavirus nsp3 phosphoprotein is essential for virus replication and functions initially within polyprotein p123 or p23 components of the short-lived minus-strand replicase, and upon polyprotein cleavage, mature nsp3 likely functions also in plus-strand synthesis. we report the identification of a second nsp3 mutant from among the a complementation group of sindbis virus (sin) heat-resistant strain, ts rna-negative mutants. the ts138 mutant possessed a change of g4303 to c, predicting an ala68- ...200314645567
sequence requirements for sindbis virus subgenomic mrna promoter function in cultured cells.the sindbis virus minimal subgenomic mrna promoter (spanning positions -19 to +5 relative to the subgenomic mrna start site) is approximately three- to sixfold less active than the fully active -98 to +14 promoter region. we identified two elements flanking the -19 to +5 region which increase its transcription to levels comparable to the -98 to +14 region. these elements span positions -40 to -20 and +6 to +14 and act synergistically to enhance transcription. nine different virus libraries were ...200111264340
arthropod-borne virus zoonosis surveillance in the cape province: 1. prospective serological investigations for virus activity in the beaufort west and middelburg districts during addition to the routine sero-epidemiological surveillance for arthropod-borne viral zoonoses in the cape province carried out by the department of medical microbiology and state health department of virology laboratory, we conducted a prospective serological investigation for virus activity during 1981 in two districts of the province, namely the beaufort west and middelburg districts, which experienced heavy rainfall during the first two months of that year. the approach used was to obtain p ...19852987499
detection of alphaviruses in a genus-specific antigen capture enzyme immunoassay using monoclonal antibodies.a genus-specific antigen capture assay using similar combinations of monoclonal antibodies for capture and detection of 24 alphaviruses belonging to the seven serocomplexes was developed. the sensitivity of the test ranged from 10(3.4) 50% tissue culture infective doses/ml for o'nyong-nyong virus to 10(6.1) 50% tissue culture infective doses/ml for middelburg virus. the antigen capture test uses a combination of cross-reacting monoclonal antibodies directed against the nucleocapsid protein and e ...19911847149
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