bluuebunny/crossref_metadata_2025_split huggingface dataset:
The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative time-course study using TaqMan RT–PCR
Authors: Alexandersen Soren , Oleksiewicz Martin B. , Donaldson Alex I. 
Published: August 2015

Foot-and-mouth disease (FMD) is a highly contagious, economically important virus disease of cloven-hoofed animals. The objective of the present study was to examine the early pathogenesis of FMD in pigs by a quantitative time-course study. Under experimental conditions, recipient pigs were infected by contact with donor pigs affected by FMD. Every 24 h from day 1 to day 4 after exposure, two recipient pigs were selected randomly, killed and necropsied. A range of tissues were analysed by a quantitative TaqMan RT–PCR method and by titration of FMD virus on primary bovine thyroid cells. The titres of virus determined by assay in cell culture and calculated from the quantitative TaqMan data correlated strongly (r>0·9), thereby establishing the validity of the TaqMan calculations. The data indicated that the replication of virus in the lungs contributes only in small part to airborne virus excretion. Sites in the pharynx, trachea and nasal mucosa are probably more important in that regard. The sites of earliest virus infection and possibly replication in recipient pigs appeared to be in the pharynx (soft palate, tonsil and floor of pharynx). The data indicated that FMD virus replication in pigs is rapid and that the majority of virus amplification occurs in the skin. A model for the progression of infection is proposed, indicating initial spread from the pharyngeal region, through regional lymph nodes and via the blood to epithelial cells, resulting in several cycles of virus amplification and spread.
Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’
Authors: Donnelly Michelle L. L. , Luke Garry , Mehrotra Amit , Li Xuejun , Hughes Lorraine E. , Gani David , Ryan Martin D. 
Published: August 2015

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.
The genome of herpesvirus of turkeys: comparative analysis with Marek’s disease viruses
Authors: Kingham Brewster F. , Zelnık Vladimır , Kopáček Juraj , Majerčiak Vladimır , Ney Erik , Schmidt Carl J. 
Published: August 2015

The complete coding sequence of the herpesvirus of turkeys (HVT) unique long (UL) region along with the internal repeat regions has been determined. This allows completion of the HVT nucleotide sequence by linkage to the sequence of the unique short (US) region. The genome is approximately 160 kbp and shows extensive similarity in organization to the genomes of Marek’s disease virus serotypes 1 and 2 (MDV-1, MDV-2) and other alphaherpesviruses. The HVT genome contains 75 ORFs, with three ORFs present in two copies. Sixty-seven ORFs were identified readily as homologues of other alphaherpesvirus genes. Seven of the remaining eight ORFs are homologous to genes in MDV, but are absent from other herpesviruses. These include a gene with similarity to cellular lipases. The final, HVT-unique gene is a virus homologue of the cellular NR-13 gene, the product of which belongs to the Bcl family of proteins that regulate apoptosis. No other herpesvirus sequenced to date contains a homologue of this gene. Of potential significance is the absence of a complete block of genes within the HVT internal repeat that is present in MDV-1. These include the pp38 and meq genes, which have been implicated in MDV-1-induced T-cell lymphoma. By implication, other genes present in this region of MDV-1, but missing in HVT, may play important roles in the different biological properties of the viruses.
A comparison of the solution structures of tobacco rattle and tobacco mosaic viruses from Raman optical activity
Authors: Blanch Ewan W. , Robinson David J. , Hecht Lutz , Barron Laurence D. 
Published: August 2015

Vibrational Raman optical activity (ROA) spectra of tobacco rattle virus (TRV) and tobacco mosaic virus (TMV) were measured and compared with a view to obtaining new information about the coat protein subunit structure of TRV. A sharp strong positive band observed at ∼1344 cm−1 in the ROA spectra of the two viruses is evidence that both contain a significant amount of a hydrated form of α-helix, but more in TRV than in TMV. Although the ROA spectrum of TMV shows significant positive intensity in the range ∼1297–1312 cm−1 characteristic of α-helix in a hydrophobic environment, as expected from the helix interface residues in the four-helix bundles that constitute the basic motif of the TMV coat protein fold, that of TRV shows little positive ROA intensity here. Instead TRV shows a strong positive ROA band at ∼1315 cm−1, of much greater intensity than bands shown here by TMV, that is characteristic of polyproline II (PPII) helix. This suggests that the additional long central and C-terminal sequences of the TRV coat proteins contain a significant amount of PPII structure, plus perhaps some β-strand judging by a prominent sharp negative ROA band shown by TRV at ∼1236 cm−1, but little α-helix. The open flexible hydrated nature of PPII helical structure is consistent with the earlier suggestions that the additional sequences are exposed and, together with a larger amount of hydrated α-helix, could serve to fill the extra volume required by the larger diameter of the cylindrical TRV particles relative to those of TMV.
Comparative pathogenesis of Helicoverpa zea S nucleopolyhedrovirus in noctuid larvae
Authors: Washburn Jan O. , Wong James F. , Volkman Loy E. 
Published: August 2015

We used a recombinant of Helicoverpa zea S nucleopolyhedrovirus containing the hsp70/lacZ reporter cassette (HzSNPV-hsp70/lacZ) to quantify mortality relationships and to elucidate early pathogenesis in two permissive hosts, Heliothis virescens and Helicoverpa zea, and one semi-permissive host, Trichoplusia ni. Fourth instar T. ni were highly resistant to fatal infection both by oral injection of occlusions and by intrahaemocoelic injection of budded virus, indicating the presence of both midgut and systemic mechanisms of resistance. In bioassays, newly moulted (40) H. zea were significantly more susceptible than 40
H. virescens to fatal infection, but mortality levels were the same for larval cohorts inoculated 16 h after the moult (416). Developmental resistance was stronger in H. zea and in both hosts, partially reversed by administration of the optical brightener M2R. In both species, developmental resistance was correlated with a reduced ability of HzSNPV to establish and/or maintain primary midgut infections. In time-course experiments using a dosage of 15 occlusions (∼LD90), lacZ expression marking the onset of primary and secondary infection was first observed in midgut columnar and tracheal cells at 4 and 12 h, respectively. Inoculation of 40 larvae resulted in approximately twofold more foci in H. zea larvae than in H. virescens, but H. zea larvae sloughed infected midgut cells at a faster rate. For both heliothines, interaction of occlusion-derived virus with primary cellular targets within the midgut epithelium was critical to the outcome of infection and a key process underlying acquisition of developmental resistance.
Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro
Authors: Carrier Karma , Xiang Yu , Sanfaçon Hélène 
Published: August 2015

The proteinase ofTomato ringspot virus(genusNepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using anin vitroprocessing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q301/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.
Quasispecies nature of the genome of Grapevine fanleaf virus
Authors: Naraghi-Arani Pejman , Daubert Steve , Rowhani Adib 
Published: August 2015

Genetic diversity was characterized in 14 isolates ofGrapevine fanleaf virus(GFLV) recovered from grapevine (Vitis vinifera). Virions were collected by immunocapture, and a 1557 bp fragment containing part of the viral coat protein gene and part of the untranslated region to its 3′ side was amplified by RT–PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and by sequencing. TheAvaII-generated RFLP patterns from the various isolates were highly variable. The isolates were passaged inChenopodium quinoa. The RFLP patterns altered with passage through the alternate host, but the variation stabilized after a number of passages. Individual genomes were recovered by cloning. The subcloned sequences were found to vary from each other by as much as 13%, and the encoded amino acid sequences by as much as 9%. The data suggest that the GFLV genome consists of quasispecies populations.
Complete nucleotide sequence and genome organization of Grapevine fleck virus
Authors: Sabanadzovic Sead , Ghanem-Sabanadzovic Nina Abou , Saldarelli Pasquale , Martelli Giovanni P. 
Published: August 2015

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3′-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5′ and 3′ ends, respectively. ORF 1 potentially encodes a 215·4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24·3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3′ end of the viral genome and encode proline-rich proteins of 31·4 kDa (p31) and 15·9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.
Vaccinia virus semaphorin A39R is a 50–55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model
Authors: Gardner Jeremy D. , Tscharke David C. , Reading Patrick C. , Smith Geoffrey L. 
Published: August 2015

Vaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytesin vitro. In this study, we show that theA39Rgene of VV strain Copenhagen (COP) encodes a 50–55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including anA39Rdeletion mutant of VV COP and a WR mutant containing theA39Rsequence from COP. Loss of the gene from COP did not affect virus growthin vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COPA39Rby VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.
The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus
Authors: Hinton Tracey M. , Crabb Brendan S. 
Published: August 2015

Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem–loop structures (H–L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189–920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem–loops H–L as well as four additional upstream stem–loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869–880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.
Characterization and complete nucleotide sequence of Strawberry mottle virus: a tentative member of a new family of bipartite plant picorna-like viruses
Authors: Thompson J. R. , Leone G. , Lindner J. L. , Jelkmann W. , Schoen C. D. 
Published: August 2015

An isolate of Strawberry mottle virus (SMoV) was transferred from Fragaria vesca to Nicotiana occidentalis and Chenopodium quinoa by mechanical inoculation. Electron micrographs of infected tissues showed the presence of isometric particles of approximately 28 nm in diameter. SMoV-associated tubular structures were also conspicuous, particularly in the plasmodesmata of C. quinoa. DsRNA extraction of SMoV-infected N. occidentalis yielded two bands of 6·3 and 7·8 kbp which were cloned and sequenced. Gaps in the sequence, including the 5′ and 3′ ends, were filled using RT–PCR and RACE. The genome of SMoV was found to consist of RNA1 and RNA2 of 7036 and 5619 nt, respectively, excluding a poly(A) tail. Each RNA encodes one polyprotein and has a 3′ non-coding region of ∼1150 nt. The polyprotein of RNA1 contains regions with identities to helicase, viral genome-linked protein, protease and polymerase (RdRp), and shares its closest similarity with RNA1 of the tentative nepovirus Satsuma dwarf virus (SDV). The polyprotein of RNA2 displayed some similarity to the large coat protein domain of SDV and related viruses. Phylogenetic analysis of the RdRp region showed that SMoV falls into a separate group containing SDV, Apple latent spherical virus, Naval orange infectious mottling virus and Rice tungro spherical virus. Given the size of RNA2 and the presence of a long 3′ non-coding region, SMoV is more typical of a nepovirus, although atypically for a nepovirus it is aphid transmissible. We propose that SMoV is a tentative member of an SDV-like lineage of picorna-like viruses.
Proteolytic processing of a human astrovirus nonstructural protein
Authors: Kiang David , Matsui Suzanne M. 
Published: August 2015

To analyse the activity of the putative 3C-like serine protease encoded in open reading frame (ORF)-1a of human astrovirus serotype 1 (HAstV-1), ORF-1a was transcribed and translated in vitro. Translation products, identified by immunoprecipitation with specific antibodies against recombinant C-terminal ORF-1a fragments, include the full-length 101 kDa (p101) protein and a 38 kDa band (p38). In addition, a 64 kDa protein (p64) was immunoprecipitated by an anti-FLAG antibody when a FLAG epitope was inserted at the N terminus of the ORF-1a product. Mutation of the amino acids predicted to form the catalytic triad of the HAstV-1 3C-like serine protease (Ser-551, Asp-489, His-461) resulted in undetectable levels of p38, supporting the involvement of the HAstV-1 3C-like serine protease and the importance of these amino acids in the processing of p101 into p38 and p64. N-terminal deletions of up to 420 aa of p101 that did not involve the predicted 3C-like serine protease motif did not alter levels of p38 compared to wild-type. C-terminal deletion analysis localized p38 to the C terminus of ORF-1a. Mutation of the P1 residue of the predicted cleavage site, which is conserved among known human and sheep astrovirus sequences, resulted in no detectable p38, supporting cleavage at the Gln-567↓Thr-568 dipeptide. These results suggest that p101 is cleaved into an N-terminal p64 fragment and a C-terminal p38 product at Gln-567↓Thr-568 in a process that is dependent on the viral 3C-like serine protease.
Identification of VP19 and VP15 of white spot syndrome virus (WSSV) and glycosylation status of the WSSV major structural proteins
Authors: van Hulten Mariëlle C. W. , Reijns Martin , Vermeesch Angela M. G. , Zandbergen Fokko , Vlak Just M. 
Published: August 2015

White spot syndrome virus (WSSV) infects penaeid shrimp and other crustaceans. The WSSV virion consists of an enveloped rod-shaped nucleocapsid enclosing a large circular double-stranded DNA genome of 293 kbp. The virion envelope contains two major proteins of 28 (VP28) and 19 kDa (VP19) and the nucleocapsid consists of three major proteins of 26 (VP26), 24 (VP24) and 15 kDa (VP15). Study on the morphogenesis of the WSSV particle requires the genomic identification and chemical characterization of these WSSV virion proteins. An internal amino acid sequence of envelope protein VP19 was obtained by amino acid sequencing and used to locate the VP19 open reading frame of this protein on the genome, as WSSV ORF182. VP19 contained two putative transmembrane domains, which may anchor this protein in the WSSV envelope. Similarly, the gene for VP15 was located on the WSSV genome as ORF109. N-terminal amino acid sequencing on VP15 suggested that this protein was expressed from the second ATG of its ORF and the first methionine is lost by N-terminal protein processing. The 15 kDa protein is very basic and is a candidate DNA-binding protein in the WSSV nucleocapsid. None of the five major structural WSSV proteins appear to be glycosylated, which is an unusual feature among enveloped animal viruses.
Analysis of transcription of Porcine circovirus type 1
Authors: Mankertz Annette , Hillenbrand Bernd 
Published: August 2015

Porcine circovirus type 1 (PCV1) contains two major open reading frames encoding the replication initiator proteins, Rep and Rep′, and the structural protein, Cap. The promoters of these two genes (P
cap
and P
rep
) have been mapped. P
cap
is located within the rep open reading frame (nt 1328–1252). P
rep
has been mapped to the intergenic region immediately upstream of the rep gene (nt 640–796) and overlaps the origin of replication of PCV1. Although binding of both rep gene products to a fragment containing P
rep
and the overlapping origin of replication has been reported, only the full-length Rep protein repressed P
rep
, while the spliced isoform Rep′ did not. P
rep
repression is mediated by binding of the Rep protein to the two inner hexamers, H1 and H2, located in the origin of PCV1, whereas binding of Rep to hexamers H3 and H4 was not necessary. Use of Rep mutants indicated that the conserved rolling-circle replication domain II as well as the P loop are essential for repression of P
rep
. In contrast to P
rep
, transcription of P
cap
was not influenced by viral proteins. Additionally, the ratio of the rep and rep′ transcripts was analysed. Twelve hours after transfection of PK15 cells with an infectious clone of PCV1, similar amounts of both transcripts were detected, but later the amount of the two transcripts varied, indicating a balanced expression of the two rep transcripts.