A conserved secondary structure in the hypervariable region at the 5' end of Bamboo mosaic virus satellite RNA is functionally interchangeable

Satellite RNA (satRNA) associated with Bamboo mosaic virus (BaMV) is dependent on BaMV for replication and encapsidation. Molecular analyses of total RNA extracted from bamboo species collected worldwide revealed that 26 out of 61 BaMV isolates harbored satBaMV. Among them, two phylogenetically distinguishable groups, A and B, with a genetic diversity of 6.9 +/- 0.7% were identified. Greatest sequence diversity occurred in the 5' untranslated region (UTR) that contained one hypervariable region with variations of up to 20.7%. Concurrent covariations in the 5' hypervariable sequences support the existence of a conserved apical hairpin stem-loop structure, which was earlier mapped by enzymatic probings and functional analyses [Annamalai, P., Hsu, Y.H., Liu, Y.P., Tsai, C.H., Lin, N.S., 2003. Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311 (1), 229-239]. Furthermore, chimeric satBaMVs generated by interchanging the hypervariable region between groups A and B demonstrated the replication competence of satBaMV isolates in Nicotiana benthamiana protoplasts co-inoculated with BaMV RNA. The results suggest that an evolutionarily conserved secondary structure exists in the hypervariable region of 5' UTR of satBaMV.     

Structural and mutational analyses of cis-acting sequences in the 5'-untranslated region of satellite RNA of bamboo mosaic potexvirus

The satellite RNA of Bamboo mosaic virus (satBaMV) contains on open reading frame for a 20-kDa protein that is flanked by a 5'-untranslated region (UTR) of 159 nucleotides (nt) and a 3'-UTR of 129 nt. A secondary structure was predicted for the 5'-UTR of satBaMV RNA, which folds into a large stem-loop (LSL) and a small stem-loop. Enzymatic probing confirmed the existence of LSL (nt 8-138) in the 5'-UTR. The essential cis-acting sequences in the 5'-UTR required for satBaMV RNA replication were determined by deletion and substitution mutagenesis. Their replication efficiencies were analyzed in Nicotiana benthamiana protoplasts and Chenopodium quinoa plants coinoculated with helper BaMV RNA. All deletion mutants abolished the replication of satBaMV RNA, whereas mutations introduced in most of the loop regions and stems showed either no replication or a decreased replication efficiency. Mutations that affected the positive-strand satBaMV RNA accumulation also affected the accumulation of negative-strand RNA; however, the accumulation of genomic and subgenomic RNAs of BaMV were not affected. Moreover, covariation analyses of natural satBaMV variants provide substantial evidence that the secondary structure in the 5'-UTR of satBaMV is necessary for efficient replication.     

Production of haemolysis and its correlation with enterotoxicity in Aeromonas spp.

A total of 147 clinical and environmental isolates of Aeromonas that included 14 A. hydrophila, 60 A. sobria and 73 A. caviae strains was tested for haemolysin production and its correlation with enterotoxicity; 108 isolates produced beta-haemolysis. For A. hydrophila and A. sobria, titres of haemolysin were 16-128 HU/ml and for A. caviae, 16-64 HU/ml. In the ileal loop test, 82 (55.8%) strains of Aeromonas spp. produced enterotoxin. Of the beta-haemolytic strains, 72.7% of A. hydrophila, 58.6% of A. sobria and 68.6% of A. caviae isolates caused fluid accumulation in rabbit ileal loops. One strain each of alpha-haemolytic A. sobria and A. caviae, one of non-haemolytic A. sobria and nine of non-haemolytic A. caviae also caused a secretory response. The beta-haemolytic strains caused significantly more (p < 0.05) fluid accumulation than the alpha- and non-haemolytic isolates regardless of their species designation. The remaining 65 (44.2%) isolates belonging to the three species included alpha-, beta- and non-haemolytic strains: they failed to cause fluid accumulation in the initial experiments but did so after one to three consecutive passages through rabbit ileal loops. Two alpha- and 13 non-haemolytic strains switched to production of beta-haemolysis when they showed positive ileal loop reactions. However, on repeated subcultures or on storage in the laboratory, all of them reverted to their original haemolytic character and no longer produced enterotoxic activity.     

3'-Terminal RNA secondary structures are important for accumulation of tomato bushy stunt virus DI RNAs

The plus-strand RNA genome of tomato bushy stunt virus (TBSV) contains a 351-nucleotide (nt)-long 3'-untranslated region. We investigated the role of the 3'-proximal 130 nt of this sequence in viral RNA accumulation within the context of a TBSV defective interfering (DI) RNA. Sequence comparisons between different tombusviruses revealed that the 3' portion of the 130-nt sequence is highly conserved and deletion analysis confirmed that this segment is required for accumulation of DI RNAs in protoplasts. Computer-aided sequence analysis and in vitro solution structure probing indicated that the conserved sequence consists of three stem-loop (SL) structures (5'-SL3-SL2-SL1-3'). The existence of SLs 1 and 3 was also supported by comparative secondary structure analysis of sequenced tombusvirus genomes. Formation of the stem regions in all three SLs was found to be very important, and modification of the terminal loop sequences of SL1 and SL2, but not SL3, decreased DI RNA accumulation in vivo. For SL3, alterations to an internal loop resulted in significantly reduced DI RNA levels. Collectively, these data indicate that all three SLs are functionally relevant and contribute substantially to DI RNA accumulation. In addition, secondary structure analysis of other tombusvirus replicons and related virus genera revealed that a TBSV satellite RNA and members of the closely related genus Aureusvirus (family Tombusviridae) share fundamental elements of this general structural arrangement. Thus, this secondary structure model appears to extend beyond tombusvirus genomes. These conserved 3'-terminal RNA elements likely function in vivo by promoting and/or regulating minus-strand synthesis.     

Experimental infection of rabbit ligated ileal loops with Treponema hyodysenteriae

An in vivo animal model was used to assess the enteropathogenicity of the etiological agent (Treponema hyodysenteriae) of swine dysentery. Multiple ligated ileal loops, prepared in New Zealand white rabbits, were challenged with either pathogenic (B78 and B204) or nonpathogenic (Pu) isolates of the organism. The pathogenic isolates induced the onset of intestinal fluid accumulation as early as 4 h, with maximal fluid induction at 18 h postchallenge. Gross lesions of the intestinal mucosa, observed in ileal loops of rabbits sacrificed 24 h postchallenge, were characteristic of swine dysentery. Both pathogenic isolates colonized the epithelial surface and eroded the mucosal barrier, as determined by histological and scanning electron microscopic observations. Intestinal fluid accumulation and erosion of the mucosal barrier were not observed in ileal loops exposed to the nonpathogenic isolate (Pu) or to either of the nonviable pathogenic (B78 and B204) isolates. The ability of pathogenic isolates to initiate and produce infection in rabbit ligated ileal loops, which closely resembles the disease in swine, provides a system with which to study experimental swine dysentery.     

Increased Escherichia coli enterotoxin detection after concentrating culture supernatants: possible new enterotoxin detectable in dogs but not in infant mice.

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.     

Nucleocytoplasmic export of type D simian retrovirus genomic RNA: identification of important genetic subregions and interacting cellular proteins.

The simian retrovirus (SRV) genome contains a constitutive transport element (CTE) within its 3' intergenic region (IR) that mediates the nuclear export of unspliced SRV RNA. The serogroup 2 SRV CTE is predicted to form a stable stem-loop structure containing two major internal loops exhibiting 180 degrees inverse symmetry, with loop face sequences A, A', B, and B' and additional minor internal and terminal loops. To begin the identification of potential CTE-interacting proteins and to assess structural requirements for protein interaction, we conducted RNA mobility shift assays using IR fragments that obliterated this region's known stable stem-loop structure. Using immunoblotting assays, we have determined that RNA helicase A, implicated in the nuclear export of unspliced SRV genomic RNA, does not appear to interact directly with either the complete serogroup 2 SRV 3' IR or the subregion RNAs and that formation of RNA-protein complexes is conferred by interaction with other novel proteins. UV crosslinking of RNA-protein complexes, coupled with RNase T1/A digestion, indicates that a novel protein of 120 kDa molecular weight interacts with the complete CTE or with individual subregion RNAs. Transfection analyses indicate that SRV recombinants containing A, A', B, or B' sequences forming the faces for two open loops undergo RNA export; only the complete sense CTE recombinant or a second recombinant containing two subregions in sense orientation that reconstitute the 3' two-thirds of the 3' IR, and contain only A' and B that form the faces for two terminal loops, are capable of SRV RNA export. These experiments indicate that secondary structural determinants of the 3' IR and multiple protein interactions may be important factors in the nuclear export of unspliced SRV RNA.     

Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon

Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.     

Toxin production by Vibrio mimicus strains isolated from human and environmental sources in Bangladesh.

Vibrio mimicus has recently been isolated from aquatic environments of Bangladesh. A total of 125 of 300 environmental isolates, representing various biotypes, and 19 human isolates were tested for enteropathogenicity by using several models. Less than 1% of the environmental isolates and slightly more than 10% of the clinical isolates produced cholera toxin-like toxin. A significant percentage of the environmental isolates (25%) and of the human isolates (74%) induced fluid accumulation in ligated rabbit ileal loops. One environmental strain produced heat-stable toxin-like enterotoxin, whereas all of the human isolates did not. V. mimicus strains were divided into the following three groups on the basis of their activity in various toxin assays: (i) organisms which produce a heat-labile enterotoxin immunobiologically similar to cholera toxin, (ii) organisms which produce a heat-stable enterotoxin-like toxin, and (iii) organisms whose whole-cell cultures have some activity characteristic of heat-labile toxin (e.g., fluid accumulation in ligated rabbit ileal loops and positive permeability factor) but are not positive by the GM1 enzyme-linked immunosorbent assay. One isolate from this group was able to elicit these results with cell-free culture filtrates. There was no correlation of biotype with toxic activity of V. mimicus isolates.     

Toxin（s）, Other Than Cholera Toxin, Produced by Environmental Non O1 Non O139 Vibrio cholerae

A total of 39 Vibrio cholerae non O1 non O139 strains were isolated from surface waters of different parts of Dhaka City, Bangladesh. All these strains showed lack of ctx or zot gene, as demonstrated by the PCR analysis. Eighteen representative strains were tested for enterotoxin production using a rabbit ileal loop model, of which live cells of 8 strains and culture filtrates of 6 strains produced fluid accumulation in ileal loops. However, none of them produced heat stable toxin （ST）, as detected by suckling mouse assay. On the other hand, 15% of isolates produced cytotoxin as detected by the Chinese Hamster Ovary （CHO） cell assay. Fifty times concentrated culture filtrates of the representative strains did not give any precipitin band against the anti-cholera toxin, suggesting the strains produced an enterotoxin, which is antigenically different from known cholera toxin （CT）. Eighty percent of the total isolates were found to be positive for heat labile haemolysin detected by tube method, whereas, 39% were found positive by the Christie-Atkins-Munch-Petersen （CAMP） method. However, 87% of the isolates were positive for haemagglutinin/protease and all of the strains were positive for mannose-sensitive-haemagglutinin assay.     

Mapping of secondary structure of the spacer region within the 5'-untranslated region of the coxsackievirus B3 RNA: possible role of an apical GAGA loop in binding La protein and influencing internal initiation of translation

Translation initiation of the coxsackievirus B3 (CVB3) RNA has been shown to be mediated by a highly ordered structure of the 5'-UTR, which harbors an internal ribosome entry site (IRES). In this study, we have investigated the 48S ribosome assembly site and also characterized the intervening spacer region between the cryptic AUG591 and the initiator AUG742. The ribosomal complex formation was mapped by toe-printing experiment using rabbit reticulocyte lysate, which showed a major toe print at nucleotide U570 corresponding to the 48S ribosome assembly site at the putative Shine-Dalgarno like sequence. Elucidation of the secondary structure of a segment encompassing the ribosome binding site and the downstream spacer region by nuclease probing and chemical modifications demonstrated distinct stem and loop structure. Interestingly, a GAGA loop in the apical region of stem-loop H was found to be phylogenetically conserved as a GNRA-loop among the coxsackie B viruses. Deletion or substitution mutation of this apical GAGA loop drastically reduced binding with human La protein and significantly affected the IRES function. The study revealed important insights into the possible role of the intervening spacer region in cellular protein binding and influencing internal initiation of translation of CVB3 RNA.     

Analysis ofCis-Acting Elements in the 5′ Leader Sequence of Alfalfa Mosaic Virus RNA 3

The leader sequence of RNA 3 of the Leiden isolate of alfalfa mosaic virus strain 425 consists of 345 nucleotides (nt) and contains four putative stem–loop structures each with a motif in the loop that resembles the internal control region 2 (ICR2) of tRNA genes. The sequence of the 5′ terminal 112 nt of this leader contains one of these stem–loop structures and is sufficient for a reduced accumulation of RNA 3 in protoplasts and a delayed accumulation in plants (E. A. G. van der Vossenet al., Nucleic Acids Res.21, 1361–1367 (1993). A number of mutations were made in this 112-nt leader sequence to investigate its role in RNA 3 accumulation. Deletion of nucleotides 23–43, 44–90, or 55–112 and inversion or duplication of nucleotides 44–90 all abolished RNA 3 accumulation. Similarly, two base substitutions in the ICR2 motif (nucleotides 60–77) abolished RNA 3 accumulation. Mutations in the stem sections of the putative stem–loop structure had various effects on RNA 3 accumulation and supported the notion that this structure is important for plus-strand promoter activity.     

Enterotoxic activity of hemolysin BL from Bacillus cereus.

Bacillus cereus causes exotoxin-mediated diarrheal food poisoning. Hemolysin BL (HBL) is a well-characterized B. cereus toxin composed of three components (B, L1, and L2) that together possess hemolytic, cytotoxic, dermonecrotic, and vascular permeability activities. Here, we show that HBL causes fluid accumulation in ligated rabbit ileal loops at a dose of 5 micrograms of each component per loop. Maximal fluid responses occurred for combinations of all three components at > or = 25 micrograms of each component per loop. Individual components and binary combinations did not cause significant fluid accumulation at 25 micrograms of each component. Specific antisera to HBL components inhibited the fluid accumulation response of crude culture supernatant from B. cereus F837/76. These antisera were tested against an antiserum to a partially characterized multicomponent diarrheal toxin described previously by Thompson et al. (N. E. Thompson, M. J. Ketterhagen, M. S. Bergdoll, and E. J. Shantz, Infect. Immun. 43:887-894, 1984). Immunoblot and immunoprecipitation analyses indicate that HBL and that toxin are identical. These results confirm previous speculation that HBL is a tripartite enterotoxin that, as for all of its other known activities, requires all three components for maximal activity.     

A Defective RNA Associated with Bamboo Mosaic Virus and the Possible Common Mechanisms for RNA Recombination in Potexviruses

A naturally occurring 1.1 kb RNA was isolated from purified virions of bamboo mosaic potexvirus isolate S (BaMV-S). This RNA is a defective RNA (D RNA) derived from a single internal deletion of the BaMV genome. A cDNA clone representing the complete nucleotide sequence of the BaMV-S D RNA was generated and its nucleotide sequence was determined. The BaMV D cDNA is 1015 nts in length [excluding the poly(A) tail] and consists of two regions corresponding to 867 nts of the 5 terminus and 148 nts of the 3 terminus of the BaMV genomic RNA. BaMV D cDNA contains a single open reading frame (ORF) encoding a putative 29.7 kDa protein comprised of a fusion of the first 258 amino acids of BaMV ORF 1 and the last 2 amino acids of coat protein. The coding capacity of D RNA was verified by in vitro translation of native BaMV-S D RNA and of 1.1 kb RNA transcribed in vitro from the full-length D cDNA. BaMV D RNA can be reproducibly generated by serial passages of BaMV-S in Nicotiana benthamiana and is the first D RNA in the potexvirus group shown to be generated de novo. Alignments of sequences surrounding the 5 and 3 junction borders of reported potexvirus D RNAs reveal a 65.2–84.6% sequence identity, suggesting that common mechanisms for viral RNA recombination are involved in the generation of potexvirus D RNAs.     

Salmonella-induced cell death is not required for enteritis in calves

Salmonella enterica serovar Typhimurium causes cell death in bovine monocyte-derived and murine macrophages in vitro by a sipB-dependent mechanism. During this process, SipB binds and activates caspase-1, which in turn activates the proinflammatory cytokine interleukin-1beta through cleavage. We used bovine ileal ligated loops to address the role of serovar Typhimurium-induced cell death in induction of fluid accumulation and inflammation in this diarrhea model. Twelve perinatal calves had 6- to 9-cm loops prepared in the terminal ileum. They were divided into three groups: one group received an intralumen injection of Luria-Bertani broth as a control in 12 loops. The other two groups (four calves each) were inoculated with 0.75 x 10(9) CFU of either wild-type serovar Typhimurium (strain IR715) or a sopB mutant per loop in 12 loops. Hematoxylin and eosin-stained sections were scored for inflammation, and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells were detected in situ. Fluid accumulation began at 3 h postinfection (PI). Inflammation was detected in all infected loops at 1 h PI. The area of TUNEL-labeled cells in the wild-type infected loops was significantly higher than that of the controls at 12 h PI, when a severe inflammatory response and tissue damage had already developed. The sopB mutant induced the same amount of TUNEL-positive cells as the wild type, but it was attenuated for induction of fluid secretion and inflammation. Our results indicate that serovar Typhimurium-induced cell death is not required to trigger an early inflammatory response and fluid accumulation in the ileum.     

Mutations in the BC‐loop of the BKV VP1 region do not influence viral load in renal transplant patients

The reactivation and replication of the BK polyomavirus (BKV) leading to BKV‐associated nephropathy (BKVAN) is one of the major complications in renal transplantation patients. BKV isolates were classified into four subtypes (I‐IV) based on genotype variations within the VP1‐coding region. The type‐specific amino acid differences cluster within the BC‐loop of the major capsid protein VP1. As demonstrated in vitro, mutations in this region also play a role in the infectivity, attachment and stability of viral particles. Therefore, we analyzed the prevalence of BC‐loop mutations in isolates of kidney transplant patients and compared their viral load in the urine. The VP1 subtyping regions of BKV isolates obtained from urine samples of 45 renal transplant patients were sequenced. The phylogenetic analysis of these sequences revealed that subtype I (66.67%) is the most prevalent genotype. The remaining isolates belong to subtype IV (33.33%). A high frequency of changes to specific amino acids within the BC‐loop was identified among the BKV isolates from renal transplant patients. Patients with BKVAN exhibited a higher viral replication than patients without nephropathy. Although titers of isolates of subtype I were higher than titers of subtype IV isolates, the difference did not reach statistical significance. In addition, amino acid changes in the BC‐loop did not influence the viral load and the incidence of BKVAN. These in vivo results demonstrate that high replication rates which serve as a predictive marker for BKVAN are not caused by altered receptor binding or affinity via mutated BC‐loops. J. Med. Virol. 81:75–81, 2009. © 2008 Wiley‐Liss, Inc.