Simultaneous determination of six penicillins in cows'' raw milk by a multiresidue high-performance liquid chromatographic method

The complete nucleotide sequence of the genomic RNA of cymbidium mosaic potexvirus (CymMV) was determined to be 6227 nucleotides in length, excluding the poly (A) tail at the 3' terminus. Similar to other potexviruses, its genome organisation is comprised of five major open reading frames (ORFs 1 to 5), encoding a Mr 160 KDa putative RNA-dependent RNA polymerase (RdRp); a Mr 26KDa/13KDa/10KDa triple-gene-block (TGB) and a Mr 24 KDa coat protein. The CymMV encoded proteins shared a high degree of homology to their corresponding proteins of other members of the potexvirus group. The nucleotide sequence of the 5' noncoding region (NCR) of CymMV and all other potexviruses initiates with GAAAA. CymMV possesses the shortest 5' NCR among all potexviruses. Based on phylogenetic comparisons of RdRp and coat protein, CymMV shares a close relationship to PAMV, NMV, WClMV and SMYEaV. This is believed to be the first record of the complete nucleotide sequence of CymMV.     

Spontaneous and engineered deletions in the 3' noncoding region of tick-borne encephalitis virus: construction of highly attenuated mutants of a flavivirus

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3' NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3'-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3' NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3' NCR and never extended into the conserved 3'-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3'-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.     

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

Sequencing and characterization of the coat protein and 3' non-coding region of a new sweet potato potyvirus

A cDNA library was constructed from viral genomic RNA purified from sweet potato plants affected by "Sweet Potato Chlorotic Dwarf disease" in an attempt to clarify the etiology of this viral complex in Argentina. By sequence analysis, some of the obtained clones were found to belong to sweet potato feathery mottle potyvirus (SPFMV), to a closterovirus and to a new potyvirus. A cDNA clone of 1,103 bp representing the coat protein cistron and 3' non-coding region of the newly identified potyvirus was further characterized. The sequence contained an ORF of 855 nucleotides with a coding capacity of 285 amino acids, followed by a 3' untranslated tail of 248 nucleotides. The core and C-terminal regions have sequences well conserved among potyviruses. Furthermore, amino acid sequence comparisons of the capsid protein with those of other described potyviruses showed 63% homology with SPFMV, 68 to 70% with two different isolates of sweet potato latent potyvirus (SPLV), 57% with sweet potato G potyvirus (SPGV) and 73% with potato virus Y (PVY). These data allowed us to propose the inclusion of this virus as a new member of the family Potyviridae, genus Potyvirus with the designation sweet potato mild speckling potyvirus (SPMSV).     

The nucleotide sequence and genome organization of mushroom bacilliform virus: a single-stranded RNA virus of Agaricus bisporus (Lange) Imbach

Mushroom bacilliform virus (MBV) is found in association with spherical virus-like particles in cultivated mushrooms (Agaricus bisporus) afflicted with La France disease. MBV possesses a monopartite ssRNA genome of positive sense and differs from the majority of characterized mycoviruses, which contain segmented dsRNA genomes. We have cloned and sequenced the MBV genome and determined that its length is 4009 nucleotides. The MBV genome contains four major and three minor open reading frames and has 5' and 3' noncoding regions of 60 and 250 nucleotides, respectively. The putative RNA-dependent RNA polymerase and the coat protein display homology with corresponding proteins encoded by certain plant viruses, particularly luteoviruses and carmoviruses.     

Nucleotide sequence of the coat protein coding region of the potyvirus tobacco vein-banding mosaic virus

The sequence of the 3' 1184 nucleotides of tobacco vein-banding mosaic virus (TVBMV) genome has been determined. It contains a single open reading frame which encompasses the whole of the coat protein of TVBMV. The sequence of the first 20 amino acids at the N-terminal region of the coat protein has also been determined chemically to be GDDQTVDAGKNVQSNQKQRN. The sequence matches the translation product of the open reading frame starting with amino acid-271; a glycine residue. Thus the coat protein of TVBMV has a calculated M(r) of 30,210. The 3' non-coding region of TVBMV is 185 nucleotides in length. Sequence alignment of the coat proteins or the 3' non-coding regions from TVBMV and other reported potyviruses indicated that TVBMV is a separate species of the potyvirus genus.     

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.     

RNA binding properties of core protein of the flavivirus Kunjin

Kunjin virus (KUN) C is a typical flavivirus core protein which is truncated in vivo to a mature form of 105 residues enriched in lysine and arginine. In order to study the possible association of KUN C with RNA in vitro, we prepared several recombinant C proteins with specific deletions, each fused at the amino-terminus to glutathione-S-transferase (GST) and expressed in E. coli. They were reacted with KUN RNA probes transcribed in vitro from cDNA representing the 5' untranslated region (5' UTR, 93 to 96 nucleotides), the 3' UTR (624 nucleotides), and the 5' UTR plus most of the C coding region (5' core, 440 nucleotides). Fusion protein C107 (incorporating mature C) bound strongly to all KUN RNA probes with apparent specificity, being completely resistant to inhibition by 800 mM NaCl, and to competition by a large excess of tRNA. In reactions with labelled KUN RNA probes putative binding sites were identified in the isolated amino-terminal (32 residues) and carboxy-terminal (26 residues) basic amino acid domains; this binding was strongly competed by unlabelled KUN UTR probes but weakly or not at all by tRNA. These small domains probably acted co-operatively in binding of mature C to KUN RNA probes. The KUN RNA-core protein binding reactions are similar to those reported with other viral coat or capsid proteins and viral RNAs.     

Antisense oligonucleotide inhibition of hepatitis C virus gene expression in transformed hepatocytes

Genetic and biochemical studies have provided convincing evidence that the 5' noncoding region (5' NCR) of hepatitis C virus (HCV) is highly conserved among viral isolates worldwide and that translation of HCV is directed by an internal ribosome entry site (IRES) located within the 5' NCR. We have investigated inhibition of HCV gene expression using antisense oligonucleotides complementary to the 5' NCR, translation initiation codon, and core protein coding sequences. Oligonucleotides were evaluated for activity after treatment of a human hepatocyte cell line expressing the HCV 5' NCR, core protein coding sequences, and the majority of the envelope gene (E1). More than 50 oligonucleotides were evaluated for inhibition of HCV RNA and protein expression. Two oligonucleotides, ISIS 6095, targeted to a stem-loop structure within the 5' NCR known to be important for IRES function, and ISIS 6547, targeted to sequences spanning the AUG used for initiation of HCV polyprotein translation, were found to be the most effective at inhibiting HCV gene expression. ISIS 6095 and 6547 caused concentration-dependent reductions in HCV RNA and protein levels, with 50% inhibitory concentrations of 0.1 to 0.2 microM. Reduction of RNA levels, and subsequently protein levels, by these phosphorothioate oligonucleotides was consistent with RNase H cleavage of RNA at the site of oligonucleotide hybridization. Chemically modified HCV antisense phosphodiester oligonucleotides were designed and evaluated for inhibition of core protein expression to identify oligonucleotides and HCV target sequences that do not require RNase H activity to inhibit expression. A uniformly modified 2'-methoxyethoxy phosphodiester antisense oligonucleotide complementary to the initiator AUG reduced HCV core protein levels as effectively as phosphorothioate oligonucleotide ISIS 6095 but without reducing HCV RNA levels. Results of our studies show that HCV gene expression is reduced by antisense oligonucleotides and demonstrate that it is feasible to design antisense oligonucleotide inhibitors of translation that do not require RNase H activation. The data demonstrate that chemically modified antisense oligonucleotides can be used as tools to identify important regulatory sequences and/or structures important for efficient translation of HCV.     

Nuclear medicine approaches for detection of axillary lymph node metastases

Rhopalosiphum padi virus (RhPV) is an aphid virus that has been considered a member of the Picornaviridae based on physicochemical properties. The 10,011-nt polyadenylated RNA genome of RhPV was completely sequenced. Analysis of the sequence revealed the presence of two open reading frames (ORFs). The predicted amino acid sequence of ORF1, representing the first 6600 nt of the RhPV genome, showed significant similarity to the nonstructural proteins of several plant and animal RNA viruses. Direct sequence analysis of the RhPV capsid proteins showed that ORF2, which represents the last 2900 nt, encodes the three structural proteins (28, 29, and 30 kDa). The predicted amino acid sequence of ORF2 is very similar to the corresponding regions of Drosophila C virus, Plautia stali intestine virus, and to a partial sequence from the 3' end of the cricket paralysis virus genome. The site of initiation of protein synthesis for ORF2 could not be determined from the amino acid and nucleotide sequences. ORF1 is preceded by 579 nt of noncoding RNA and the two ORFs are separated by more than 500 nt of noncoding RNA. Like picornaviruses, these regions may function to facilitate the cap-independent initiation of translation of the two ORFs. These data suggest that RhPV, Drosophila C virus, Plautia stali intestine virus, and probably cricket paralysis virus are members of a unique group of small RNA viruses that infect primarily insects.     

Bowel obstruction in the postoperative period of laparoscopic inguinal hernia repair (TAPP): review of the literature

The complete nucleotide sequence of the gene 4 of rice yellow stunt rhabdovirus (RYSV) was determined from cDNAs corresponding to the viral genomic RNA. Gene 4 is 913 nucleotides (nt) long, comprising a 17-nt untranslated 5' region, a 786-nt open reading frame encoding a polypeptide with a molecular mass of 29,125 Da, and a 110-nt untranslated 3' region. Western blot analysis of the RYSV proteins using the antiserum raised against the protein expressed from the cloned gene in Escherichia coli indicates that gene 4 encodes the M protein of RYSV. Comparisons of the deduced amino acid sequence of the M protein of RYSV with those of other rhabdoviruses revealed no significant homologies. However, it shared a similar basic property and a similar distribution of charges with the other rhabdovirus matrix proteins and showed a relatively closer relationship to the sonchus yellow net virus (SYNV) M1 protein.     

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.     

Tobamovirus evolution: gene overlaps, recombination, and taxonomic implications

Tobamoviruses, mostly isolated from solanaceous plants, may represent ancient virus lineages that have codiverged with their hosts. Recently completed nucleotide sequences of six nonsolanaceous tobamoviruses allowed assessment of the codivergence hypothesis and support a third subgroup within tobamoviruses. The genomic sequences of 12 tobamoviruses and the partial sequences of 11 others have been analyzed. Comparisons of the predicted protein sequences revealed three clusters of tobamoviruses, corresponding to those infecting solanaceous species (subgroup 1), those infecting cucurbits and legumes (subgroup 2), and those infecting crucifers. The orchid-infecting odontoglossum ringspot tobamovirus was associated with subgroup 1 genomes by its coat and movement protein sequences, but with the crucifer-pathogenic tobamoviruses by the remainder of its genome, suggesting that it is the progeny of a recombinant. For four of five genomic regions, subgroup 1 and 3 genomes were equidistant from a subgroup 2 genome chosen for comparison, suggesting uniform rates of evolution. A phylogenetic tree of plant families based on the tobamoviruses they harbor was congruent with that based on rubisco sequences but had a different root, suggesting that codivergence was tempered by rare events of viruses of one family colonizing another family. The proposed subgroup 3 viruses probably have an origin of virion assembly in the movement protein gene, a large (25-codon) overlap of movement and coat protein open reading frames, and a comparably shorter genome. Codon-position-dependent base compositions and codon prevalences suggested that the coat protein frame of the overlap region was ancestral. Bootstrapped parsimony analysis of the nucleotides in the overlap region and of the sequences translated from the -1 frame (the subgroup 3 movement protein frame) of this region produced trees inconsistent with those deduced from other regions. The results are consistent with a model in which a no or short overlap organization was ancestral. Despite encoding of subgroup 2 and 3 movement protein C-termini by nonhomologous nucleotides, weak similarities between their amino acid sequences suggested convergent sequence evolution.     

Expression of glucose transporters in rat brain following transient focal ischemic injury

To better understand genetic diversity of mammalian reoviruses, we studied sequence variability in the S3 gene segment of 17 field-isolate reovirus strains and prototype strains of the three reovirus serotypes. Strains studied were isolated over a 37-year period from different mammalian hosts and geographic locations. A high degree of variability was observed in the nucleotide sequences of the S3 gene, whereas the deduced amino acid sequences of the S3 gene product, sigma NS, were highly conserved. When variability among the S3 nucleotide sequences was analyzed using pairwise comparisons, we found that 5' and 3' noncoding regions were significantly more conserved than the remainder of the gene. This high degree of sequence conservation was also observed within the first 15 nucleotides of the 5' coding region. Phylogenetic analyses showed that multiple alleles of the S3 gene cocirculate and that genetic diversity in the S3 gene does not correlate with host species, geographic locale, or date of isolation. Phylogenetic trees constructed from variation in the S3 sequences are distinct from those previously generated from sequences that encode attachment protein sigma 1, core protein sigma 2, and outer capsid protein sigma 3, which supports the hypothesis that reovirus gene segments reassort in nature. These findings suggest that reovirus gene segments are well-adapted to mammalian hosts and that reovirus evolution has reached an equilibrium.     

Poly (rC) binding protein 2 forms a ternary complex with the 5'-terminal sequences of poliovirus RNA and the viral 3CD proteinase

Poly(rC) binding protein 2 (PCBP2) forms a specific ribonucleoprotein (RNP) complex with the 5'-terminal sequences of poliovirus genomic RNA, as determined by electrophoretic mobility shift assay. Mutational analysis showed that binding requires the wild-type nucleotide sequence at positions 20-25. This sequence is predicted to localize to a specific stem-loop within a cloverleaf-like secondary structure element at the 5'-terminus of the viral RNA. Addition of purified poliovirus 3CD to the PCBP2/RNA binding reaction results in the formation of a ternary complex, whose electrophoretic mobility is further retarded. These properties are consistent with those described for the unidentified cellular protein in the RNP complex described by Andino et al. (Andino R, Rieckhof GE, Achacoso PL, Baltimore D, 1993, EMBO J 12:3587-3598). Dicistronic RNAs containing mutations in the 5' cloverleaf-like structure of poliovirus that abate PCBP2 binding show a decrease in RNA replication and translation of gene products directed by the poliovirus 5' noncoding region in vitro, suggesting that the interaction of PCBP2 with these sequences performs a dual role in the virus life cycle by facilitating both viral protein synthesis and initiation of viral RNA synthesis.