Biomedical subjects

A Michael Lindberg

Publications and source records attributed to A Michael Lindberg.

14 recordsLinked to original sources

PubMed2006

Molecular analysis of duck hepatitis virus type 1 reveals a novel lineage close to the genus Parechovirus in the family Picornaviridae.

Duck hepatitis virus type 1 (DHV-1) was previously classified as an enterovirus, based primarily on observed morphology and physicochemical properties of the virion. The complete nucleotide sequences of two strains (DRL-62 and R85952) of DHV-1 have been determined. Excluding the poly(A) tail, the genomes are 7691 and 7690 nt, respectively, and contain a single, large open reading frame encoding a polyprotein of 2249 aa. The genome of DHV-1 is organized as are those of members of the family Picornaviridae: 5' untranslated region (UTR)-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' UTR. Analysis of the genomic and predicted polyprotein sequences revealed several unusual features, including the absence of a predicted maturation cleavage of VP0, the presence of two unrelated 2A protein motifs and a 3' UTR extended markedly compared with that of any other picornavirus. The 2A1 protein motif is related to the 2A protein type of the genus Aphthovirus and the adjacent 2A2 protein is related to the 2A protein type present in the genus Parechovirus. Phylogenetic analysis using the 3D protein sequence shows that the two DHV-1 strains are related more closely to members of the genus Parechovirus than to other picornaviruses. However, the two DHV-1 strains form a monophyletic group, clearly distinct from members of the genus Parechovirus.

3' Untranslated Regions↗

PubMed2005

A novel and rapid method to quantify cytolytic replication of picornaviruses in cell culture.

Determining viral titers is a key issue in a wide variety of studies regarding different aspects of virology. The standard methods used for determining picornavirus titers are endpoint titration assay and plaque assay, both time consuming and laborious. The method described uses the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide) that is reduced to formazane by cellular dehydrogenase, genes shown to be down-regulated during picornavirus infection. The amount formazane produced correlates with the viral titers obtained and can easily be measured using an ELISA plate reader. The colorimetric method has been evaluated using virus types from different genera of the Picornaviridae family. The MTT method reduces the time spent on determining the viral titers and still maintains a reliable accuracy.

Animals↗

PubMed2005

Cytolytic replication of coxsackievirus B2 in CAR-deficient rhabdomyosarcoma cells.

The six coxsackievirus B serotypes (CVB1-6) use the coxsackie- and adenovirus receptor (CAR) for host cell entry. Four of these serotypes, CVB1, 3, 5 and 6, have also shown the capacity to replicate and cause cytolysis in rhabdomyosarcoma (RD) cells, a CAR-deficient cell line. This extended tropism has been associated with an acquired ability to bind decay accelerating factor (DAF). In this study, we have adapted the CVB2 prototype strain Ohio-1 (CVB2/O) to replicate in RD cells. Two types of infection were identified: (I) an enterovirus-typical, lytic infection, and (II) a non-lytic infection. Both CVB2/O-RD variants retained the prototype-ability to cause cytopathic effect in HeLa cells using CAR as receptor. Phenotypic and genotypic changes in the CVB2/O-RD-variants were determined and compared to the prototype cultured in HeLa cells. Inhibition studies using antibodies against CAR and DAF revealed a maintained ability of the CVB2/O-RD-variants to bind CAR, but no binding to DAF was observed. In addition, neither the prototype nor the CVB2/O-RD-variants were able to cause hemagglutination in human red blood cells, an enterovirus feature associated with affinity for DAF. Sequence analysis of the CVB2/O-RD-variants showed acquired mutations in the capsid region, suggesting extended receptor usage towards an alternative, yet unidentified, receptor for CVB2.

Adaptation, Physiological↗

PubMed2005

Oncolysis of vascular malignant human melanoma tumors by Coxsackievirus A21.

Cultured melanoma cell lines despite exhibiting similar in vitro morphology, display significant phenotypic and growth rate differences when propagated as in vivo xenografts. Previously we have shown that Coxsackievirus A21 (CVA21) lytically infects in vitro cultures of malignant melanoma cells and is efficient at reducing the tumor burden of mice bearing slow-growing SK-Mel-28 melanoma xenografts. The oncolytic activity of CVA21 against in vivo melanoma xenografts, which possess rapid growth rates and more extensive vascular structure than SK-Mel-28 xenografts warrants further investigation. In the present study we evaluated the oncolytic action of CVA21 against rapidly growing melanoma xenografts (ME4405) which exhibit a highly vascular phenotype. Flow cytometric analysis indicated that in vitro cultures of ME4405 cells expressed comparable levels of the CVA21 cellular receptors, ICAM-1 (intercellular adhesion molecules-1) and DAF (decay accelerating factor) to SK-Mel-28 cells. Despite similar levels of CVA21 receptor expression, SK-Mel-28 cells appear to be more susceptible to viral lysis than ME4405 cells, even though the kinetics of virus replication in both lines was comparable. Intratumoral, intraperitoneal or intravenous administration of CVA21 were equally effective in reducing the tumor volume of ME4405 xenografts in immunodeficient mice, and provides further evidence for the use of CVA21 as a novel oncolytic agent against varying phenotypes of malignant melanoma.

Animals↗

PubMed2004

Cell culture propagation and biochemical analysis of the Ljungan virus prototype strain.

Ljungan virus (LV) is proposed as a potentially important rodent harbored viral human pathogen. Little is known about the biophysical nature of the virus and despite being molecularly characterized, progress in epidemiological and basic biological studies of LV has been hampered by the lack of a robust and reliable cell culture propagation system. Here we report the first description of an efficient lytic multi-cycle cell culture propagation of the LV prototype strain (87-012). Biophysical analysis of gradient purified LV virions generated by this system identified mature infectious virions to possess a sedimentation coefficient of 160S and in agreement with previous molecular prediction, polyprotein analysis suggests that the native virion is composed of only three major structural proteins. The nucleotide composition of the complete genome of the LV cell culture adapted virus was determined and compared to that of the parental prototype LV. Numerous mutations were observed scattered throughout the viral genome and particularly in VP1. The development of this cell culture system for LV should open new avenues in the study of LV biology, structure, pathogenesis, and prevalence of natural infection in the wider community.

Animals↗

PubMed2004

Analysis of the serotype and genotype correlation of VP1 and the 5' noncoding region in an epidemiological survey of the human enterovirus B species.

The sequence identity of the enterovirus VP1 gene has been shown to correlate with the serotype concept. Enterovirus molecular typing methods are therefore often based on sequencing of the VP1 genomic region and monophyletic clustering of VP1 sequences of a homologous serotype. For epidemiological surveillance, 342 enterovirus samples obtained from patients with aseptic meningitis in Belgium from 1999 to 2002 were first diagnosed as being enterovirus positive by amplification of the 5' noncoding region (5'NCR) by reverse transcription (RT)-PCR. Subsequently, samples were molecularly typed by RT-nested PCR amplification and sequencing of a portion of the VP1 gene. Phylogenetic analyses were performed to investigate enteroviral evolution and to examine the serotype and genotype correlation of the two genomic regions. Our typing results demonstrated echovirus 30, echovirus 13, echovirus 18, and echovirus 6 to be the most predominant types. Echoviruses 13 and 18 were considered to be emerging human serotypes since 2000 and 2001, respectively, as they had been rarely reported before. Several serotypes existed as multiple genotypes (subtypes) from 1999 to 2002, but genomic differences mainly resided at synonymous sites; these results strongly suggest that the subtypes exhibit similar antigenic properties. Phylogenetic analyses confirmed that VP1 is an adequate region for molecular typing. Serotype-specific clusters are not observed commonly in phylogenetic trees based on the 5'NCR, and the phylogenetic signal in the 5'NCR was found to be particularly low. However, some substructure in the 5'NCR tree made a tentative prediction of the enterovirus type possible and was therefore helpful in PCR strategies for VP1 (e.g., primer choice), provided some background knowledge on the local spectrum of enteroviruses already exists.

Base Sequence↗

PubMed2004

Enterovirus capsid interactions with decay-accelerating factor mediate lytic cell infection.

The cellular receptor usage of numerous human enteroviruses can differ significantly between low-cell-culture-passaged clinical isolates and highly laboratory-passaged prototype strains. The prototype strain of coxsackievirus A21 (CVA21) displays a dual-receptor specificity as determined with a receptor complex consisting of decay-accelerating factor (DAF) and intercellular adhesion molecule 1 (ICAM-1). In this study, the cellular receptor interactions of low-cell-passage CVA21 clinical isolates with respect to their interactions with cell surface-expressed DAF and ICAM-1 were compared to those of the CVA21 prototype (Kuykendall) strain. Dual-receptor usage of DAF and ICAM-1 by CVA21 clinical isolates was confirmed by cell transfection and radiolabeled binding assays. The cellular attachment of clinical and prototype CVA21 strains to cells that coexpressed DAF and ICAM-1 was not additive compared to the viral binding to cells expressing one or other receptor. In fact, the binding data suggest there is an inhibition of CVA21 cellular attachment in environments where high-level coexpression of both DAF and ICAM-1 occurs. Antibody cross-linking of DAF rendered cells susceptible to lytic infection by the CVA21 clinical isolates. In a novel finding, three clinical isolates could, to various degrees, infect and lyse DAF-expressing cells in the absence of DAF-antibody cross-linking and ICAM-1 expression. Sequence analysis of the P1 region of clinical and prototype virus genomes identified a number of coding changes that may contribute to the observed enhanced DAF usage phenotype of the clinical CVA21 isolates. None of the amino acid changes was located in the previously postulated ICAM-1 footprint, a receptor-binding environment that was conserved on the capsid surface of all CVA21 clinical isolates. Taken together, the data suggest that community-circulating strains of CVA21 can infect target cells expressing either ICAM-1 or DAF alone and that such interactions extend tissue tropism and impact directly on viral pathogenesis.

Adult↗

PubMed2003

Molecular typing and epidemiology of enteroviruses identified from an outbreak of aseptic meningitis in Belgium during the summer of 2000.

Non-polio enteroviruses are the most common cause of aseptic meningitis worldwide. From May to September 2000, a major outbreak of aseptic meningitis occurred in Belgium. Cerebrospinal fluid samples (CSF) of 122 patients were found to contain enterovirus RNA using diagnostic RT-PCR that targeted a 231-bp gene fragment in the 5' noncoding region. In addition, a molecular typing method was developed based on RT-nested PCR and sequencing directly from CSF(a) 358-bp fragment in the aminoterminal part of the VP1 capsid protein. To identify the enterovirus type, nucleotide sequences of the VP1 amplicons were compared to all the enterovirus VP1 sequences available in GenBank. Echovirus 30 (31.2%), echovirus 13 (23.8%), and echovirus 6 (20.5%) were identified most frequently during the epidemic. Coxsackievirus B5 was present in 15.6% of the samples, and could be subdivided in two distinct epidemic clusters, coxsackievirus B5a (10.7%) and B5b (4.9%). Other enteroviruses encountered were echovirus 16 (5.7%), echovirus 18 (1.6%), coxsackievirus B4 (0.8%) and echovirus 7 (0.8%). The high prevalence of echovirus 13, considered previously a rare serotype, indicates it is an emerging epidemic type. To verify the typing results and to explore further the intratypical genetic variation, phylogenetic analysis was carried out. Geographical clustering of most of the strains within each type and subtype could be observed. The RT-nested PCR strategy, carried out directly on clinical samples, is a simple and rapid method for adequate molecular typing of the Group B enteroviruses causing aseptic meningitis.

Adolescent↗

PubMed2003

Molecular characterization of M1146, an American isolate of Ljungan virus (LV) reveals the presence of a new LV genotype.

Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles in Sweden. This study describes the genetic characterization of a virus, M1146, which was isolated in 1962 from another vole species (Microtus montanus), trapped in Oregon, USA. Based on antigenic properties, M1146 was postulated previously as a putative member of the family PICORNAVIRIDAE: The near complete genomic sequence verifies that M1146 is a member of the Picornaviridae, most closely related to LVs isolated in Sweden. The strain M1146 possesses typical LV genomic organization, including a cluster of two 2A homologues. There are significant differences throughout the capsid protein region, while the non-structural region of M1146 is closely related to the Swedish LV genomes. Genetic and phylogenetic analyses show that M1146 represents a new genotype within the distinct LV cluster. Isolation of LV from both Swedish and American voles trapped over a period of 30 years suggests a continuous worldwide presence.

Animals↗

PubMed2003

Evolution of the genome of Human enterovirus B: incongruence between phylogenies of the VP1 and 3CD regions indicates frequent recombination within the species.

Enteroviruses show a high degree of sequence variation both between and within serotypes due to the lack of proofreading of the viral RNA-dependent RNA polymerase. In addition, recombination is known to occur not only within but also between different serotypes. We have previously shown that capsid coding sequences of coxsackievirus B4 (CVB4) cluster in several coexisting genotypes (intergenotypic nucleotide difference of 12 % or more) whereas a single lineage of echovirus 30 (EV30) has been prevailing and evolving throughout the last two decades. In the major capsid gene, VP1, clustering of both nucleotide and amino acid sequences correlates with serotype. We have now determined a 501 nucleotide sequence in the non-structural 3CD region of CVB4 and EV30 field strains. Phylogenetic analysis revealed that sequences of Human enterovirus B (HEV-B) were segregated in the 3CD region into three distinct clusters without the VP1-associated serotype/genotype correlation. One of the clusters comprised the E2 strain of CVB4, the EV30 prototype and five other CVB4 field strains whereas the other two clusters, in addition to CVB4 and EV30 strains, also included other HEV-B serotypes. We believe that intertypic recombination is the most likely explanation for the observed incongruence. Similarity analysis based on complete genomes of the CVB4 and EV30 prototypes and the CVB4 E2 strain revealed that a putative recombination spot was mapped within the 2B gene. The incongruence observed in the two genomic domains (P1 and P3) suggests a certain degree of independent evolution, which may be explained by interserotypic recombination within an enterovirus species. It is thus difficult to exclude recombination in the history of any given strain.

Animals↗

PubMed2003

Cellular receptor interactions of C-cluster human group A coxsackieviruses.

The cellular receptor complex of coxsackievirus A21 (CVA21), a C-cluster human enterovirus, is formed by the subtle interaction of individual cellular receptors, decay accelerating factor (DAF) and intercellular adhesion molecule-1 (ICAM-1). In this receptor complex, DAF functions in the membrane sequestration of the virus, while the role of ICAM-1 is as the functional cellular internalization receptor. However, despite the elucidation of the CVA21-cell receptor interactions, there have been few definite investigations into cellular receptor usage of other coxsackie A viruses (CVAs) belonging to the C-cluster. In the present study, radiolabelled virus-binding assays demonstrated that CVA13, -15, -18 and -20, a subset of the human enterovirus C-cluster, bind directly to surface-expressed ICAM-1, but not to surface-expressed DAF. Furthermore, lytic infection of ICAM-1-expressing rhabdomyosarcoma (RD) cells by this C-cluster subset of viruses was inhibited by specific ICAM-1 monoclonal antibody blockade, except for that of CVA20. Despite possessing ICAM-1-binding capabilities, CVA20 employed an as yet unidentified internalization receptor for cell entry and subsequent productive lytic infection of ICAM-1-negative RD cells. In a further example of C-cluster cellular receptor heterogeneity, CVA13 exhibited significant binding to the surface of CHO cells expressing neither DAF nor ICAM-1. Despite a common receptor usage of ICAM-1 by this subset of C-cluster CVAs, the amino acid residues postulated to represent the ICAM-1-receptor footprint were not conserved.

Amino Acid Sequence↗

PubMed2002

Molecular analysis of three Ljungan virus isolates reveals a new, close-to-root lineage of the Picornaviridae with a cluster of two unrelated 2A proteins.

Ljungan virus (LV) is a suspected human pathogen recently isolated from bank voles (Clethrionomys glareolus). In the present study, it is revealed through comparative sequence analysis that three newly determined Swedish LV genomes are closely related and possess a deviant picornavirus-like organization: 5' untranslated region-VP0-VP3-VP1-2A1-2A2-2B-2C-3A-3B-3C-3D-3' untranslated region. The LV genomes and the polyproteins encoded by them exhibit several exceptional features, such as the absence of a predicted maturation cleavage of VP0, a conserved sequence determinant in VP0 that is typically found in VP1 of other picornaviruses, and a cluster of two unrelated 2A proteins. The 2A1 protein is related to the 2A protein of cardio-, erbo-, tescho-, and aphthoviruses, and the 2A2 protein is related to the 2A protein of parechoviruses, kobuviruses, and avian encephalomyelitis virus. The unprecedented association of two structurally different 2A proteins is a feature never previously observed among picornaviruses and implies that their functions are not mutually exclusive. Secondary polyprotein processing of the LV polyprotein is mediated by proteinase 3C (3C(pro)) possessing canonical affinity to Glu and Gln at the P1 position and small amino acid residues at the P1' position. In addition, LV 3C(pro) appears to have unique substrate specificity to Asn, Gln, and Asp and to bulky hydrophobic residues at the P2 and P4 positions, respectively. Phylogenetic analysis suggests that LVs form a separate division, which, together with the Parechovirus genus, has branched off the picornavirus tree most closely to its root. The presence of two 2A proteins indicates that some contemporary picornaviruses with a single 2A may have evolved from the ancestral multi-2A picornavirus.

3' Untranslated Regions↗

PubMed2001

Genetic characterization of the coxsackievirus B2 3' untranslated region.

The secondary structure of the 3' untranslated region (3'UTR) of picornaviruses is thought to be important for the initiation of negative-strand RNA synthesis. In this study, genetic and biological analyses of the 3' terminus of coxsackievirus B2 (CVB2), which differs from other enteroviruses due to the presence of five additional nucleotides prior to the poly(A) tail, is reported. The importance of this extension was investigated using a 3'UTR mutant lacking the five nucleotides prior to the poly(A) tail and containing two point mutations. The predicted secondary structure within the 3'UTR of this mutant was less energetically favourable compared with that of the wild-type (wt) genotype. This mutant clone was transfected into green monkey kidney cells in four parallel experiments and propagated for multiple passages, enabling the virus to establish a stable revertant genotype. Genetic analysis of the virus progeny from these different passages revealed two major types of revertant. Both types showed wt-like growth properties and more stable and wt-like predicted secondary structures than the parent mutant clone. The first type of revertant neutralized the introduced point mutation with a compensatory second-site mutation, whereas the second type of revertant partly compensated for the deletion of the five proximal nucleotides by the insertion of nucleotides that matched the wt sequence. Therefore, the extended 3' end of CVB2 may be considered to be a stabilizing sequence for RNA secondary structure and an important feature for the virus.

3' Untranslated Regions↗

PubMedDate not supplied

Development of type 1 diabetes in wild bank voles associated with islet autoantibodies and the novel ljungan virus.

Wild bank voles (Clethrionomys glareolus) may develop diabetes in laboratory captivity. The aim of this study was to test whether bank voles develop type 1 diabetes in association with Ljungan virus. Two groups of bank voles were analyzed for diabetes, pancreas histology, autoantibodies to glutamic acid decarboxylase (GAD65), IA-2, and insulin by standardized radioligand-binding assays as well as antibodies to in vitro transcribed and translated Ljungan virus antigens. Group A represented 101 trapped bank voles, which were screened for diabetes when euthanized within 24 hours of capture. Group B represented 67 bank voles, which were trapped and kept in the laboratory for 1 month before being euthanized. Group A bank voles did not have diabetes. Bank voles in group B (22/67; 33%) developed diabetes due to specific lysis of pancreatic islet beta cells. Compared to nondiabetic group B bank voles, diabetic animals had increased levels of GAD65 (P < .0001), IA-2 (P < .0001), and insulin (P = .03) autoantibodies. Affected islets stained positive for Ljungan virus, a novel picorna virus isolated from bank voles. Ljungan virus inoculation of nondiabetic wild bank voles induced beta-cell lysis. Compared to group A bank voles, Ljungan virus antibodies were increased in both nondiabetic (P < .0001) and diabetic (P = .0015) group B bank voles. Levels of Ljungan virus antibodies were also increased in young age at onset of newly diagnosed type 1 diabetes in children (P < .01). These findings support the hypothesis that the development of type 1 diabetes in captured wild bank voles is associated with Ljungan virus. It is speculated that bank voles may have a possible zoonotic role as a reservoir and vector for virus that may contribute to the incidence of type 1 diabetes in humans.

Adolescent↗