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P P Mertens

Publications and source records attributed to P P Mertens.

At least 19 recordsLinked to original sources
PubMed2001

Translocation portals for the substrates and products of a viral transcription complex: the bluetongue virus core.

The bluetongue virus core is a molecular machine that simultaneously and repeatedly transcribes mRNA from 10 segments of viral double-stranded RNA, packaged in a liquid crystalline array. To determine how the logistical problems of transcription within a sealed shell are solved, core crystals were soaked with various ligands and analysed by X-ray crystallography. Mg(2+) ions produce a slight expansion of the capsid around the 5-fold axes. Oligonucleotide soaks demonstrate that the 5-fold pore, opened up by this expansion, is the exit site for mRNA, whilst nucleotide soaks pinpoint a separate binding site that appears to be a selective channel for the entry and exit of substrates and by-products. Finally, nucleotides also bind to the outer core layer, providing a substrate sink.

Base Sequence↗
PubMed2001

Sequence characterization of Ndelle virus genome segments 1, 5, 7, 8, and 10: evidence for reassignment to the genus Orthoreovirus, family Reoviridae.

The full-length nucleotide sequences of genome segments 1, 5, 7, 8 and 10 from Ndelle virus (NDEV) have been characterized. Comparison of the deduced protein amino acid sequences with those of other member viruses of the family Reoviridae demonstrates that NDEV was originally assigned incorrectly to the genus Orbivirus (aa identity values of <20%). In contrast, high levels of amino acid identity were found with members of the species Mammalian orthoreovirus (MRV); for example, amino acid identity in gamma3(Pol) is between 91 and 97%. These findings, together with previous antigenic analyses, provide evidence that NDEV should be reclassified as a new serotype (designated MRV-4) within the Mammalian orthoreovirus species.

Genome, Viral↗
PubMed1999

The highly ordered double-stranded RNA genome of bluetongue virus revealed by crystallography.

The concentration of double-stranded RNA within the bluetongue virus core renders the genome segments liquid crystalline. Powder diffraction rings confirm this local ordering with a 30 A separation between strands. Determination of the structure of the bluetongue virus core serotype 10 and comparison with that of serotype 1 reveals most of the genomic double-stranded RNA, packaged as well-ordered layers surrounding putative transcription complexes at the apices of the particle. The outer layer of RNA is sufficiently well ordered by interaction with the capsid that a model can be built and extended to the less-ordered inner layers, providing a structural framework for understanding the mechanism of this complex transcriptional machine. We show that the genome segments maintain local order during transcription.

Animals↗
PubMed1999

A comparison of six cypovirus isolates by cross-hybridisation of their dsRNA genome segments.

Genetic relationships between the genome segments of six cypovirus (CPV) isolates were analysed by RNA cross-hybridisation. These included three type 1 viruses and single isolates of types 2, 5 and 12, which collectively are identical to those previously compared by serology and electrophoresis [Mertens et al. (1989), J Gen Virol 70: 173-185]. Since only genome segment 10 of three cypovirus types and segments 8 and 9 of a single virus strain (of type 1) have currently been sequenced, this initial study provides some additional information on sequence variation/similarity in each of the ten genome segments. The RNA of the type 1 viruses showed high levels of cross-hybridisation. Significant but much lower levels of cross-hybridisation were detected between type 1 and the related type 12 CPV. However, only very low levels of cross-hybridisation were detected between the other pairs of viruses. Apart from evidence of a slightly higher level of sequence similarity between the largest segments, the RNA sequence appeared to vary uniformly across the whole genome. There was no evidence for any type specific RNA sequences restricted to individual genome segment(s). The sequence variation, reflected in the levels of RNA sequence similarity and cross hybridisation, correlates well with serological data, showing large differences between CPV types and supports the continued use of electropherotype as one of the 'species parameters' for the classification of cypoviruses.

Animals↗
PubMed1999

The barriers to bluetongue virus infection, dissemination and transmission in the vector, Culicoides variipennis (Diptera: Ceratopogonidae).

Transmission of bluetongue virus (BTV) by a vector species of Culicoides was studied using immunohistochemistry, virus titration and in vitro transmission tests. Adult female C. variipennis were used from two colonies that are either "transmission competent" or "transmission refractory" after oral infection with BTV. Intrathoracic (i.t.) injection of BTV into the haemocoel always resulted in a fully disseminated infection and transmission of virus in saliva. However, after ingestion of an infectious blood meal, only 30% (approximately) of midges from either colony became persistently infected. Although none of the orally infected insects from the "refractory" colony were able to transmit virus, 12% of those from the "competent" colony (containing > or = 10(3.0)TCID50 of virus/midge) did transmit BTV in their saliva. The most important barriers to BTV transmission in Culicoides vector species appeared to be a mesenteron infection barrier (MIB), which controls initial establishment of persistent infection, a mesenteron escape barrier (MEB) which can restrict virus to gut cells and a dissemination barrier (DB) which can prevent virus which enters the haemocoel from infecting secondary target organs. Culicoides variipennis do not appear to present either a salivary gland infection barrier (SGIB), or a salivary gland escape barrier (SGEB) to BTV.

Animals↗
PubMed1999

Lipofectin increases the specific activity of cypovirus particles for cultured insect cells.

Cytoplasmic polyhedrosis viruses (CPV) are classified as 14 distinct species (electropherotypes) within the genus Cypovirus, family Reoviridae. Cypovirus research has been limited by a lack of appropriate cell culture systems (for each of these virus species) in which the majority of cells can become productively infected. Lipofection increased the infection rate of Lymantria dispar 652 cells, by virus particles (derived from polyhedra) of Orgyia pseudosugata type 5 cypovirus (Op-5 CPV), from 3 to 44%. Lipofection also significantly increased the percentage of Trichoplusia ni 368 cells infected with the same virus (from < 1 to approximately 7%). The spread of cypovirus infection between cells was either very slow or insignificant, and infected cells appeared to remain viable for long periods. Virus infection was detected by the observation of polyhedra formation in individual cells and it was therefore possible to develop a simple quantitative assay system to measure virus titre (TCID50). Cryo-electron microscopy showed that cypovirus particles formed a complex with the lipid, involving their envelopment within the liposome membrane. It was concluded that the increased infectivity of the virus by lipofection was due to a more efficient cell entry mechanism, probably involving fusion between liposome and cell membranes.

Animals↗
PubMed1999

The structure of a cypovirus and the functional organization of dsRNA viruses.

Cytoplasmic polyhedrosis virus (CPV) is unique among the double-stranded RNA viruses of the family Reoviridae in having a single capsid layer. Analysis by cryo-electron microscopy allows comparison of the single shelled CPV and orthoreovirus with the high resolution crystal structure of the inner shell of the bluetongue virus (BTV) core. This suggests that the novel arrangement identified in BTV, of 120 protein subunits in a so-called 'T=2' organization, is a characteristic of the Reoviridae and allows us to delineate structural similarities and differences between two subgroups of the family--the turreted and the smooth-core viruses. This in turn suggests a coherent picture of the structural organization of many dsRNA viruses.

Bluetongue virus↗
PubMed1998

Capping and methylation of mRNA by purified recombinant VP4 protein of bluetongue virus.

The core of bluetongue virus (BTV) is a multienzyme complex composed of two major proteins (VP7 and VP3) and three minor proteins (VP1, VP4, and VP6) in addition to the viral genome. The core is transcriptionally active and produces capped mRNA from which all BTV proteins are translated, but the relative role of each core component in the overall reaction process remains unclear. Previously we showed that the 76-kDa VP4 protein possesses guanylyltransferase activity, a necessary part of the RNA capping reaction. Here, through the use of highly purified (>95%) VP4 and synthetic core-like particles containing VP4, we have investigated the extent to which this protein is also responsible for other activities associated with cap formation. We show that VP4 catalyzes the conversion of unmethylated GpppG or in vitro-produced uncapped BTV RNA transcripts to m7GpppGm in the presence of S-adenosyl-L-methionine. Analysis of the methylated products of the reaction by HPLC identified both methyltransferase type 1 and type 2 activities associated with VP4, demonstrating that the complete BTV capping reaction is associated with this one protein.

Bluetongue virus↗
PubMed1998

The atomic structure of the bluetongue virus core.

The structure of the core particle of bluetongue virus has been determined by X-ray crystallography at a resolution approaching 3.5 A. This transcriptionally active compartment, 700 A in diameter, represents the largest molecular structure determined in such detail. The atomic structure indicates how approximately 1,000 protein components self-assemble, using both the classical mechanism of quasi-equivalent contacts, which are achieved through triangulation, and a different method, which we term geometrical quasi-equivalence.

Amino Acid Sequence↗
PubMed1998

Taxonomy of African horse sickness viruses.

Nine distinct genera are currently recognised within the virus family Reoviridae, which include a total of 63 virus groups or species (species = virus group = electropherotype or serogroup), comprising 214 virus serotypes or subtypes, as well as 20 provisional types or subtypes, most of which (149 + 9 tentative) are assigned to the genus Orbivirus [5, 9, 16]. The 19 species of orbiviruses (serogroups), were established principally on antigenic (serologic) grounds but many of these placements have been supported by molecular analyses. This introductory paper defines the taxonomy and classification of these viruses and establishes guidelines for use in other paper to be presented at this symposium and elsewhere.

African Horse Sickness↗
PubMed1998

VP7 from African horse sickness virus serotype 9 protects mice against a lethal, heterologous serotype challenge.

An established mouse model system was used to evaluate the effectiveness of the major outer core protein VP7 of African horse sickness virus (AHSV) serotype 9 as a subunit vaccine. Balb C mice were immunised with VP7 crystals purified from AHSV infected BHK cells. In groups of mice, each of which was immunised with > or = 1.5 micrograms of the protein in Freund's adjuvant, > or = 80% of mice survived challenge with a virulent strain of a heterologous AHSV serotype (AHSV 7), that killed > or = 80% of the mice in the uninoculated control groups. This level of protection was significantly greater than that observed in mice inoculated with equivalent amounts of either denatured VP7 (50% survival), or GST/VP7 fusion protein (50-70% survival), or which were vaccinated with AHSV 9 (40-50% survival). The VP7 protein folding, or its assembly into crystals, are thought to play some role in the effectiveness of the protective response observed. Titres of circulating antibodies against AHSV VP7 were determined by competitive ELISA but did not appear to correlate with the levels of protection observed. Passive transfer of these antibodies to syngeneic recipients also failed to protect Balb C mice from the AHSV 7 challenge. The observed protection is therefore unlikely to be due to an antibody mediated immune response.

African Horse Sickness↗
PubMed1998

Molecular epidemiology of African horse sickness virus based on analyses and comparisons of genome segments 7 and 10.

This paper describes a method to rapidly identify African horse sickness virus (AHSV), using a single tube reverse transcription polymerase chain reaction (PCR). This method was used to amplify cDNA copies of genome segments 7 and 10 from several different AHSV strains, of different serotypes, which were then analysed by sequencing and/or endonuclease digestion. AHSV VP7 (encoded by genome segment 7) is one of the two major capsid proteins of the inner capsid layer, forming the outer surface of the core particle. VP7 is highly conserved and is the major serogroup specific antigen common to all nine AHSV serotypes. Digestion of the 1179 bp cDNA with restriction enzymes, allowed differentiation of several strains of different serotypes and identified six distinct groups containing AHSV-1, 3, 6 and 8; AHSV-2; AHSV-4; AHSV-5; AHSV-7; and AHSV-9. Differences were detected between wild type viruses and vaccine strains that had been attenuated by multiple passage in suckling mouse brain or in tissue cultures. RFLP analysis was also used to study variation the 758 bp cDNA copies of AHSV genome segment 10, which encodes the two small non-structural membrane proteins NS3 and NS3a. In this way it was possible to distinguish each of the strains tested, except AHSV 4 (USDA) and AHSV 9 (USDA). However, these isolates could be distinguished by RFLP analysis of genome segment 7 cDNA. Using sequence analysis of genome segment 10 we were able to classify the virus isolates into three groups: AHSV-1, 2 and 8; AHSV-3 and 7; AHSV 4, 5, 6 and 9. These studies confirmed that the virus which first appeared in central Spain in July 1987, subsequently spread into northern Morocco in October 1989.

African Horse Sickness↗
PubMed1998

Structural studies of orbivirus particles.

We are using crystallographic methods to investigate the structure of AHSV and BTV. Our initial approach was to investigate the structure of the major protein component of the viral core, VP7(T13). This trimeric protein has been studied in several crystal forms from both orbiviruses and reveals a structure made up of conserved domains, capable of conformational changes and possessing a cleavage site. Further crystallographic analyses of native particles have provided a picture of the VP7(T13) and VP3(T2) layers of the BTV core. The VP7(T13) layer consists of 260 trimers arranged rather symmetrically and possessing very similar structures, thereby following the rules of quasi equivalence. The VP3(T2) layer is thin and contains 120 copies of 100 kDa protein arranged as 60 approximate dimers. This type of icosahedral construction has not been observed before and appears to contain a genome which is highly ordered. We anticipate that all of these features will be common to AHSV.

African Horse Sickness Virus↗
PubMed1998

Development of a mouse model system, coding assignments and identification of the genome segments controlling virulence of African horse sickness virus serotypes 3 and 8.

Attenuated (att) and wild type (wt) strains of the nine AHSV serotypes were evaluated for virulence in adult Balb C mice. Although most were avirulent in this system, isolates of AHSV 1att, 3wt, 3att, 4wt, 5att, 7att and 8att caused some mortality when administered via an intranasal route. After plaque cloning, only the attenuated vaccine strain of AHSV 7att caused any mortality via an intravenous route. AHSV 3att and AHSV 8wt were virulent (V) and avirulent (AV) (respectively) in the mouse model and were selected as parental strains for production of genome segment reassortants. These progeny virus strains were plaque cloned, then characterised to identify the genome segments that influence virulence of AHSV in the mouse model. Three virulence phenotypes were observed: fully virulent (V); fully avirulent (A); and a novel intermediate virulence (N) not expressed by either parental strain. Genome segment 2 (encoding outer capsid protein VP2) from the avirulent parent appeared to have a controlling influence in production of the A phenotype. Reassortants with the V phenotype all contained segment 2 from the virulent parent, however in each case they also contained genome segments 5 and 10, also from AHSV 3 (V). Genome segments 5 and 10 encode the smaller outer capsid protein VP5 and the non structural proteins NS3/NS3a, respectively. A combination of genome segments 2, 5 and 6 from the avirulent parent and segment 10 from the virulent parent were found in each of the virus strains with the N phenotype. However, comparison of two reassortants (A79 and A790), which differ only in a single segment, showed that replacement of genome segment 10 from the avirulent parent with that from the virulent parent, conferred the N phenotype on A790.

African Horse Sickness↗
PubMed1998

Phylogenetic analysis of African horse sickness virus segment 10: sequence variation, virulence characteristics and cell exit.

African horse sickness virus (AHSV) genome segment 10 encodes the non-structural proteins NS3/NS3a, which is involved in release of virus from cells. Full length segment 10 cDNAs were amplified by reverse transcription-polymerase chain reaction, from isolates of AHSV serotypes 2, 3, 4, 5, 7, 8 and 9. These cDNAs were cloned, sequenced and their phylogenetic relationships analysed. High levels of sequence homology were detected in segment 10 from some isolates of different serotypes, confirming that they could be grouped on this basis (serotypes 4, 5, 6 and 9 (group alpha); serotypes 3 and 7 (group beta); serotypes 1, 2, and 8 (group gamma). However, data from bluetongue virus (the prototype orbivirus) indicate that the AHSV serotype is determined exclusively by the structural outer coat proteins VP2 and VP5, encoded by genome segments 2 and 5 respectively. Therefore, as a direct consequence of genome segment reassortment between AHSV strains from different serotypes, the differences observed in segment 10 do not give a reliable indication of virus serotype. Segment 10 of AHSV 3 (virulent) and AHSV 3att (attenuated) were also analysed. These strains, together with AHSV 8, have been used to study of the genetic basis of virulence using reassortment (O'Hara et al., this publication). Virus release studies, using Culicoides cell cultures, indicate that differences in segment 10 of AHSV 3att and 8 can influence the timing of virus release from the infected cell.

African Horse Sickness Virus↗
PubMed1997

African horsesickness virus VP7 sub-unit vaccine protects mice against a lethal, heterologous serotype challenge.

An established mouse model was used to evaluate the effectiveness of the major outer core protein of African horsesickness virus (AHSV), VP7, as a subunit vaccine. Adult female BALB/c mice were immunized with VP7 crystals purified from BHK cells infected with AHSV serotype 9 (AHSV-9), using three inoculations in Freund's adjuvant. Eighty to one hundred per cent of the immunized mice were protected against a heterologous challenge with a known lethal dose of AHSV-7. The protected immunized mice did not develop any clinical signs characteristic of virulent AHSV infection in this model during the study. In contrast, 80-100% mortality was observed in the non-immunized mice that received the same challenge virus. Subsequent studies indicated that a single inoculation of 1.5 micrograms purified AHSV VP7 in Freund's complete adjuvant was sufficient to protect at least 90% of mice from AHSV-7 challenge. If the antigen was presented in the absence of Freund's complete adjuvant, 70% of the mice were still protected by one inoculation of VP7 crystals. Titres of circulating antibody against AHSV VP7, determined by competitive ELISA, did not appear to correlate with protection and passive antibody transfer from immunized BALB/c mice failed to protect syngeneic recipients from AHSV-7 challenge. Therefore, the observed protection is unlikely to be due to an antibody-mediated immune response. The number of viraemic mice and the duration of viraemia post-challenge was significantly reduced in vaccinated mice compared to non-vaccinated controls. However, the levels of viraemia were similar.

African Horse Sickness↗
PubMed1996

Expression of the major core structural protein (VP7) of bluetongue virus, by a recombinant capripox virus, provides partial protection of sheep against a virulent heterotypic bluetongue virus challenge.

A recombinant capripox virus was constructed containing a cDNA copy of genome segment 7 of bluetongue virus (BTV) serotype 1 from South Africa (BTV 1SA), which expressed high levels of the major BTV core protein VP7 in infected lamb testis (LT) cells. Sheep vaccinated with this recombinant virus developed antibodies to VP7 (detected by ELISA) but no neutralizing antibodies to either the homologous or heterologous BTV serotype, prior to challenge (BTV 1 or BTV 3, respectively). Following challenge with a virulent heterotypic strain of BTV (BTV3 SA), all of the animals developed clinical signs of disease, indicating that they were infected and that the challenge virus did replicate. While all of the control animals died, six of the eight animals that were vaccinated with the recombinant capripox virus expressing VP7 recovered fully. This is the first report of a significant level of cross serotype protection against the lethal effects of a challenge with virulent BTV, produced by vaccination with a single BTV core protein, which did not generate a neutralizing antibody response.

Animals↗