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Classification of RNA secondary structures using the techniques of cluster analysis.

An RNA molecule has a lot of suboptimal secondary structures. Some of these suboptimal structures are very similar and some are entirely different. In some cases, the free energy of these suboptimal structures does not differ much from that of the optimal structure. In order to characterize this relationship more clearly, we defined a metric among the secondary structures by the unweighted pair group method, using the arithmetic average and extended this metric to the sets of secondary structures. We report two applications of this metric. First, we developed a method for the classification of these suboptimal secondary structures of a given RNA sequence. Results of classification are presented with the secondary structures of cadang-cadang coconut viroid, potato spindle tuber viroid and polio virus as examples. Second, we applied this metric to the classification of the secondary structures derived from a set of mutant RNA sequences. We discuss that the mutation of a given RNA sequence changes not only the optimal secondary structure, but also the population of the cluster of the secondary structures.

Animals↗

Epstein-Barr virus-associated anaplastic large cell variant of diffuse large B-cell-type non-Hodgkin's lymphoma with concurrent p53 protein expression.

In the new World Health Organization (WHO) classification of malignant lymphoma, anaplastic large cell lymphoma of B-cell phenotype is classified either as the anaplastic large cell variant of diffuse large B-cell lymphoma or as Hodgkin's lymphoma. A 71-year-old Japanese man developed fever and generalized lymphadenopathy. Biopsy of the right axillary node revealed morphology of malignant lymphoma in which large cells with abundant cytoplasm and pleomorphic nuclei were scattered among small lymphocytes. Immunostaining with various monoclonal antibodies revealed the large cells to be CD79+, CD20/L26+, CD45RO/UCHL-(1-), CD3-, CD10-, CD30+, NPM/ALK-, EMA-, CD15-, and bcl-(2-). Amplification of the J region of the immunoglobulin heavy chain by polymerase chain reaction revealed a single rearranged band. Therefore the diagnosis of anaplastic large cell variant of diffuse large B-cell lymphoma, stage IIIB, was made from the standpoint of the new WHO classification of malignant lymphoma. Biopsy led to findings of Epstein-Barr virus (EBV)-associated lymphoma with positive in situ hybridization results for EBV small RNAs, positive results of immunostaining with EBV latent membrane 1 antibody, and negative results of immunostaining with Epstein-Barr nuclear antigen 2. Results of immunostaining of the mass with p53 antibody also were positive for lymphoma cells. The findings in this case may suggest a close relationship between p53 expression and latent EBV infection.

Aged↗

Human papillomaviruses and cervical neoplasia: a model for carcinogenesis.

Human papillomaviruses are etiologic for cervical cancers and their pathologic precursors. As presented in this review, pathologic, epidemiologic, and molecular data all support a working model that accounts for the pathogenetic role of these viruses in cervical neoplasia. Diagnostic criteria and classification systems are discussed in light of this model. These insights point to a potential change in clinical screening systems for cervical cancer. In addition, vaccine trials for oncogenic HPVs have begun. In the long term, these trials may hold promise as truly specific preventive therapy for this common human cancer.

Female↗

Secondary structure of the 3'-noncoding region of flavivirus genomes: comparative analysis of base pairing probabilities.

The prediction of the complete matrix of base pairing probabilities was applied to the 3' noncoding region (NCR) of flavivirus genomes. This approach identifies not only well-defined secondary structure elements, but also regions of high structural flexibility. Flaviviruses, many of which are important human pathogens, have a common genomic organization, but exhibit a significant degree of RNA sequence diversity in the functionally important 3'-NCR. We demonstrate the presence of secondary structures shared by all flaviviruses, as well as structural features that are characteristic for groups of viruses within the genus reflecting the established classification scheme. The significance of most of the predicted structures is corroborated by compensatory mutations. The availability of infectious clones for several flaviviruses will allow the assessment of these structural elements in processes of the viral life cycle, such as replication and assembly.

Algorithms↗

[Cognitive evoked potentials at the stages II and III of human immunodeficiency virus infection].

Neuropsychological and psychometric investigations have sometimes attested to and sometimes denied the existence of cognitive perturbations during the early phases of human immunodeficiency virus (HIV) infection (stages II and III of the CDC classification): strictly asymptomatic seropositivity or generalized lymphadenopathy. Therein lies the basis of the debate concerning the neurotropism of the virus and its deleterious effects on the central nervous system (CNS). We hoped to contribute to the resolution of this discussion by recording late evoked auditory potentials, which are composed of two types of components: exogenous responses attesting to the reception of stimuli by specific sensory areas of the CNS, regardless of their informative value for the subject; and endogenous responses that occur later, appearing when the subject is required to distinguish between different stimuli, for example, counting high-pitched sounds randomly distributed among low-pitched ones. The latter responses, which have been most extensively studied for wave P300, are associated with cognitive functions, and alterations of the evoked cognitive potentials have been observed during the course of demential syndromes of various origins. Fifteen individuals were subjected to the protocol for recording long-latency, evoked auditory potentials. These studies were completed by a battery of psychometric tests, two methods for evaluating depression and an assessment of the anxiety level. The results showed a significant lengthening of the latency of wave P300 in the seropositive subjects. This prolongation also affected one of the exogenous components, i.e., wave P2. In addition, their intelligence quotients, regardless of whether the IQ explores the so-called crystallized component or the fluid component of intelligence, were not significantly different from those of the general population.(ABSTRACT TRUNCATED AT 250 WORDS)

Acquired Immunodeficiency Syndrome↗

Interaction between hepatitis b virus and alcohol consumption in liver cirrhosis. An epidemiologic study.

The relationship between alcohol consumption and the presence of seric hepatitis B virus markers has been studied in 125 male patients with liver cirrhosis and 126 control subjects. Mean daily alcohol consumption was found to be higher in patients than in controls. Hepatitis B virus seric markers were also more frequently observed in patients than in controls (53.6% vs. 36.5%, 2p less than 0.05). This was due to the greater frequency of hepatitis B core antibody alone or with hepatitis B surface antigen in the patients (2p less than 0.05). The possible interaction between alcohol and B virus was studied by log-linear hierarchic models for contingency tables. Data were displayed in a three-way classification frequency table (diagnosis, daily alcohol consumption, and hepatitis B virus seric markers). A model assuming that alcohol and seric markers of unresolved infection by hepatitis B virus are independently linked to cirrhosis gives the better goodness of fit between observed and estimated frequencies. The result however, suggests, that hepatitis B virus infection does not increase the influence of alcohol consumption on the production of cirrhosis.

Adult↗

Mapping of antigenic changes in the haemagglutinin of Hong Kong influenza (H3N2) strains using a large panel of monoclonal antibodies.

A panel of 125 monoclonal antibodies (IgG) was raised against the haemagglutinin of an early representative of the Hong Kong (H3N2) subtype of influenza. They were classified into groups based on their cross-reactions with 16 other virus strains from the same subtype. This classification was performed using methods of numerical taxonomy. Statistical tests supported the validity of the grouping. Ten such groups were identified. Nine antibodies remained unclassified. The locations on the haemagglutinin molecule of amino acid residues influencing the binding of each antibody group were estimated. This was achieved by a study of antibody cross-reactive profiles, coupled with previously published locations of amino acid changes in the primary sequence of different haemagglutinins, and their positions in the tertiary structure of the molecule. The locations of the amino acids affecting antibody binding overlapped between the different antibody groups forming a continuous ring surrounding the probable cell-receptor pocket. The amino acids affecting the binding of each antibody group may or may not represent the actual antibody binding sites. The importance of the different sites of amino acid variation in the haemagglutinin during evolution of the virus is discussed.

Amino Acid Sequence↗

H5N2 avian influenza outbreak in Texas in 2004: the first highly pathogenic strain in the United States in 20 years?

In early 2004, an H5N2 avian influenza virus (AIV) that met the molecular criteria for classification as a highly pathogenic AIV was isolated from chickens in the state of Texas in the United States. However, clinical manifestations in the affected flock were consistent with avian influenza caused by a low-pathogenicity AIV and the representative virus (A/chicken/Texas/298313/04 [TX/04]) was not virulent for experimentally inoculated chickens. The hemagglutinin (HA) gene of the TX/04 isolate was similar in sequence to A/chicken/Texas/167280-4/02 (TX/02), a low-pathogenicity AIV isolate recovered from chickens in Texas in 2002. However, the TX/04 isolate had one additional basic amino acid at the HA cleavage site, which could be attributed to a single point mutation. The TX/04 isolate was similar in sequence to TX/02 isolate in several internal genes (NP, M, and NS), but some genes (PA, PB1, and PB2) had sequence of a clearly different origin. The TX/04 isolate also had a stalk deletion in the NA gene, characteristic of a chicken-adapted AIV. By analyzing viruses constructed by in vitro mutagenesis followed by reverse genetics, we found that the pathogenicity of the TX/04 virus could be increased in vitro and in vivo by the insertion of an additional basic amino acid at the HA cleavage site and not by the loss of a glycosylation site near the cleavage site. Our study provides the genetic and biologic characteristics of the TX/04 isolate, which highlight the complexity of the polygenic nature of the virulence of influenza viruses.

Animals↗

Phylogeny of the genus Flavivirus.

We undertook a comprehensive phylogenetic study to establish the genetic relationship among the viruses of the genus Flavivirus and to compare the classification based on molecular phylogeny with the existing serologic method. By using a combination of quantitative definitions (bootstrap support level and the pairwise nucleotide sequence identity), the viruses could be classified into clusters, clades, and species. Our phylogenetic study revealed for the first time that from the putative ancestor two branches, non-vector and vector-borne virus clusters, evolved and from the latter cluster emerged tick-borne and mosquito-borne virus clusters. Provided that the theory of arthropod association being an acquired trait was correct, pairwise nucleotide sequence identity among these three clusters provided supporting data for a possibility that the non-vector cluster evolved first, followed by the separation of tick-borne and mosquito-borne virus clusters in that order. Clades established in our study correlated significantly with existing antigenic complexes. We also resolved many of the past taxonomic problems by establishing phylogenetic relationships of the antigenically unclassified viruses with the well-established viruses and by identifying synonymous viruses.

Amino Acid Sequence↗

Differential interactions among isolates of peanut stunt cucumovirus and its satellite RNA.

The interactions of seven isolates of peanut stunt cucumovirus (PSV) originating from North America, Europe and Africa, and two variants of PSV satellite RNA (sat RNA) were analysed. Electrophoretic and immunoblot analyses of the coat protein (CP) and Northern blot hybridization analyses of the viral RNAs showed that isolates PSV F352, 1339 and 1507 belonged to subgroup I, and isolates PSV W, Su and B to subgroup II. The seventh isolate, robinia mosaic virus (RoMV) clustered with subgroup I isolates by CP analysis, but was related to both subgroups by RNA hybridization analysis. The ability to support the accumulation of two newly described sat RNA variants, P4 and P6 sat RNAs, was not related to PSV isolate classification: neither PSV W nor RoMV were helper viruses for these PSV sat RNAs. Symptom modulation by both sat RNAs was the same: the presence of sat RNA did not modify the symptoms induced by subgroup I isolates but exacerbated the symptoms induced by subgroup II isolates in both tobacco and cowpea. Sat RNAs P4 and P6 contained 393 nucleotides, and differed only in three nucleotide substitutions. This resulted in marked differences in infectivity, level of accumulation and relative encapsidation between both the sat RNAs. Accumulation levels and relative encapsidation of sat RNAs was also affected by the isolate of helper virus.

Arachis↗

Prevalence of hepatitis G virus RNA in French blood donors and recipients.

BACKGROUND: Recently, cases of chronic hepatitis were linked to the presence of genomic sequences of a newly described RNA virus termed hepatitis G virus (HGV) and belonging to the Flaviviridae family. STUDY DESIGN AND METHODS: The presence of HGV RNA was searched for by polymerase chain reaction in a population of blood donors and in patients who had received multiple blood component transfusions and/or intravenous immunoglobulin (IVIG) infusions. RESULTS: Twenty-one (4.2%) of 500 donors were positive for HGV RNA as were 21 (10.7%) of 196 nonimmunosuppressed patients who had received multiple transfusions of packed red cells, 4 (8.7%) of 46 common variable immune deficiency (CVID) patients who had received only IVIG, and 22 (24.7%) of 89 bone marrow transplant (BMT) patients who had received IVIG and cellular components. The proportion of HGV-positive individuals was significantly higher in the immunosuppressed recipients (CVID and BMT patients) than in the nonimmunosuppressed patients who were multiply transfused with packed red cells (p < 0.03). The proportion of HGV-positive individuals was significantly higher in the BMT patients who had received IVIG and cellular components than in the CVID patients who had received IVIG only (p < 0.03). Eight (17.0%) of the 47 HGV-positive recipients and 48 (16.9%) of the 284 HGV-negative recipients had a serum alanine aminotransferase level higher than the upper limit of normal (nonsignificant difference). The medical history of HGV-positive donors failed to reveal a particular at-risk event. The large majority of HGV-infected patients had a normal serum alanine aminotransferase level, and the proportion of patients with elevated alanine aminotransferase was the same in HGV-positive and in HGV-negative recipients. CONCLUSION: The pathological significance of HGV infection remains unelucidated, and the classification of HGV as a new hepatitis virus was perhaps premature.

Adult↗

Absence of double-stranded replicative forms of HCV RNA in liver tissue from chronically infected patients.

The mechanism of hepatitis C virus (HCV) replication is unknown, although the classification of HCV in the Flaviviridae has led to the postulation that HCV may adopt a replication strategy similar to that of the flaviviruses. To determine if HCV double-stranded replicative forms, consistent with this strategy, were present in total liver RNA extracted from HCV-infected individuals, HCV-specific RNA was detected by reverse transcription followed by polymerase chain reaction (RT-PCR). Initially, a strand-specific RT-PCR resulting from chemical modification of the 3' end of the RNA was established using in vitro transcribed HCV RNA. This procedure allowed the specific detection of positive and negative HCV RNA strands in HCV-infected liver tissue. The species of HCV RNA was then examined in RNA extracted from liver tissue from naturally infected individuals; total liver RNA was either: (i) fractionated with 2M LiCl (designed to precipitate single-stranded and partially double-stranded RNA); or (ii) digested with RNase A in high salt conditions (designed to digest single-stranded RNA only). Amplification of positive sense HCV RNA from the LiCl-insoluble fraction, but not from the LiCl-soluble fraction nor in the RNase A-digested sample, was consistent with the interpretation that single-strand, but not double-stranded HCV RNA, was contained in the liver samples. Thus, it is unclear if a double-stranded RNA species is formed during the HCV replication cycle.

Electrophoresis, Agar Gel↗

HPV6 variants from malignant tumors with sequence alterations in the regulatory region do not reveal differences in the activities of the oncogene promoters but do contain amino acid exchanges in the E6 and E7 proteins.

Human papillomavirus type 6 (HPV6) causes benign epithelial proliferations of the anogenital and aerodigestive tract, which usually tend to regress spontaneously. The low incidence of HPV6 in carcinomas and the rare progression of the benign tumors has led to the classification of HPV6 as "low-risk" virus. A series of reports, however, described the isolation of HPV6 variants from malignant tumors characterized by sequence rearrangements in the noncoding regulatory region (NCR). It was speculated that these sequence alterations play a role in tumor progression by enhancing the promoter activity and thereby increasing the expression of the viral oncogenes E6 and E7. To elucidate if HPV6 isolates from malignancies do regularly exhibit sequence alterations in the regulatory region we first determined and complied the sequences of the NCRs of a number of isolates from benign and malignant lesions. This analysis revealed in general a high degree of sequence conservation between the individual isolates. Most of the isolates, however, differed, independently of origin, by a major and one or two minor insertions from the prototype HPV6b sequence. When tested in a functional assay these altered NCR sequences did not result in significantly different activities of the promoters responsible for the expression of the E6 and E7 genes. Further analysis of the E6 and E7 coding region revealed a surprisingly high sequence variability within the E6 ORF and allowed the detection of amino acid exchanges unique for isolates from carcinomas.

Base Sequence↗

Transcription map of the African green monkey lymphotropic papovavirus.

The S1 endonuclease mapping technique was employed to characterize polyadenylated RNA molecules encoded by the African green monkey lymphotropic papovavirus (LPV) in B-lymphoblastoid cells of human origin infected with the virus. Two groups of stable transcripts of opposite polarities were identified and mapped. By analogy with other papovaviruses, these molecules were designated as "early" and "late" RNAs. The early transcripts include two RNA molecules consisting of a colinear 3' "main body" of about 1.9 kb spliced to 5' "leaders" of 0.26 and 0.60 kb, respectively. These molecules are presumably mRNAs encoding the LPV large T and small t antigens. Shorter, less abundant early RNA molecules have also been detected but have not been mapped. The late RNAs include three molecules, of which two consist of a common leader of 0.22 kb spliced to main bodies of 1.2 and 1.85 kb, respectively. The third late transcript of 2.3 kb is colinear. These late transcripts are presumably mRNAs encoding the LPV capsid proteins. A survey of the LPV DNA sequence published by M. Pawlita, A. Clad, and H. zur Hausen (1985, Virology 143, 196-211) allowed tentative assignment of the termini and splice sites of the various RNAs to nucleotides in the DNA sequence. The LPV transcription map is similar but not entirely analogous to those of polyoma virus and simian virus 40. These distinctions support a previous classification of LPV as a member of a new subgroup of the polyomaviruses.

Animals↗

A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction.

The scattering density of the virus is represented as a truncated weighted sum of orthonormal basis functions in spherical coordinates, where the angular dependence of each basis function has icosahedral symmetry. A statistical model of the image formation process is proposed and the maximum likelihood estimation method computed by an expectation-maximization algorithm is used to estimate the weights in the sum and thereby compute a 3-D reconstruction of the virus particle. If multiple types of virus particle are represented in the boxed images then multiple 3-D reconstructions are computed simultaneously without first requiring that the type of particle shown in each boxed image be determined. Examples of the procedure are described for viruses with known structure: (1). 3-D reconstruction of Flockhouse Virus from experimental images, (2). 3-D reconstruction of the capsid of Nudaurelia Omega Capensis Virus from synthetic images, and (3). 3-D reconstruction of both the capsid and the procapsid of Nudaurelia Omega Capensis Virus from a mixture of unclassified synthetic images.

Algorithms↗

HIV type 1 phenotype correlates with the stage of infection in vertically infected children.

A cohort of 39 vertically infected children (class N, A, B, and C of the CDC HIV classification for pediatric infection) was studied by virus isolation and non-syncytium inducing (NSI)/syncytium inducing (SI) HIV-1 phenotype evaluation. The HIV-1 isolates were recovered from PBMCs and the MT-2 cell line was used to perform the syncytium assay. HIV-1 could be isolated in 34 of 39 (87%) infected children, regardless of the clinical and immunological stage of the disease. Class N and A subjects harbored exclusively NSI strains, whereas the SI phenotype was detected in two of eight class B and five of nine class C patients. All of the SI variants were observed in severely CD4-depleted children (class 3 patients). The capability of pediatric HIV-1 isolates to induce a cytopathic effect is associated with the clinical status and the degree of CD4 depletion. These data suggest that the biological properties of HIV-1 isolates in children do not differ from those observed in adults, and that viral phenotype strictly correlates with disease progression in vertically infected children.

Cell Line↗

Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.

BACKGROUND: Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. PURPOSE: The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. METHODS: The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, RESULTS: The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. CONCLUSION: These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. IMPLICATIONS: It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

Adult↗