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Papers by Philip Minor

Research paper thumbnail of Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

Virology journal, 2006

The genome of coronaviruses contains structural and non-structural genes, including several so-ca... more The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV) 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU) in Salisbury, UK. All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%). Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates c...

Research paper thumbnail of Characterization of the echovirus 7 receptor: domains of CD55 critical for virus binding

Journal of virology, 1995

CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates compleme... more CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates complement activity by accelerating the decay of C3/C5 convertases. Recently, we and others have established that this molecule acts as a cellular receptor for echovirus 7 and related viruses. DAF consists of five domains: four short consensus repeats (SCRs) and a serine/threonine-rich region, attached to the cell surface by a glycosylphosphatidyl inositol anchor. Chinese hamster ovary cells stably transfected with deletion mutants of DAF or DAF-membrane cofactor protein recombinants were analyzed for virus binding. The results indicate that the binding of echovirus 7 to DAF specifically requires SCR2, SCR3, and SCR4. There is also a nonspecific requirement for the S/T-rich region which probably functions to project the binding region away from the cell membrane. The three nonpeptide modifications of DAF, N-linked glycosylation, O-linked glycosylation, and the glycosylphosphatidyl inositol ancho...

Research paper thumbnail of Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus

Journal of virology, 1995

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from i... more Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensit...

Research paper thumbnail of Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses

European journal of biochemistry / FEBS, Jan 3, 1983

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of... more Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.

Research paper thumbnail of Intertypic genomic rearrangements of poliovirus strains in vaccinees

Virology, 1988

In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus i... more In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus isolates from a normal asymptomatic primary vaccinee. This study examines isolates from additional primary vaccinees to determine whether intertypic recombination is a general occurrence in excreted polioviruses. T1 RNase oligonucleotide finger-printing and limited dideoxy primer extension RNA sequencing demonstrated no evidence of intertypic recombination among type 1 or type 2 excreted strains. However, vaccinees excreting type 3 strains for long periods of time eventually produced recombinant strains involving either type 1 or type 2 poliovirus. Moreover, a characteristic time course of appearance of excreted type 3 intertypic recombinant polioviruses was established. Type 3/type 1 and type 3/type 2 recombinant strains appeared at Days 10-11 with a single crossover site in the gene for nonstructural protein 2C. Type 3/type 2/type 3 complex recombinant strains with an additional crossov...

Research paper thumbnail of The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity

Virology, 1991

Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been gene... more Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved.

Research paper thumbnail of The Complete Nucleotide Sequence of Coxsackievirus B4 and Its Comparison to Other Members of the Picornaviridae

Journal of General Virology, 1987

SUMMARY The genome of the prototype strain of coxsackievirus B4 (J.V.B. Benschoten) has been clon... more SUMMARY The genome of the prototype strain of coxsackievirus B4 (J.V.B. Benschoten) has been cloned in Escherichia coil and its complete nucleotide sequence determined. Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids. The predicted amino acid sequence of the polyprotein shows close homology (88~) to

Research paper thumbnail of Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

Research paper thumbnail of No confirmation of pestivirus RNA in human virus vaccines

Journal of Clinical Microbiology

Recently, Harasawa and Tomiyama (1) reported the detection of pestivirus RNA in human vaccines. T... more Recently, Harasawa and Tomiyama (1) reported the detection of pestivirus RNA in human vaccines. The origin of the contamination was unknown, but the vaccines had been prepared in cell cultures supplemented with bovine fetal calf serum (FCS), which can be contaminated with bovine viral diarrhea virus (BVDV).

Research paper thumbnail of Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

Journal of Virological Methods, 2015

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultan... more Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate full genome coverage. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

Research paper thumbnail of Antigenic and Molecular Properties of Type 3 Poliovirus Responsible for an Outbreak of Poliomyelitis in a Vaccinated Population

Journal of General Virology, 1986

SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serological... more SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing

Research paper thumbnail of Isolation of SV40 from the environment of a colony of cynomolgus monkeys naturally infected with the virus

Virology, 2004

The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected ... more The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected with this virus has been analyzed by testing waste of the cage samples.

Research paper thumbnail of Current challenges in implementing cell-derived influenza vaccines: implications for production and regulation, July 2007, NIBSC, Potters Bar, UK

Vaccine, Jan 14, 2009

A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use o... more A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use of cell culture systems for the manufacture of vaccines against influenza. Issues discussed included the effect of using eggs and different cell types in strain selection, development of seed viruses to be used in production and the nature of the reagents to be used in determining vaccine potency. Future studies to progress the field were reviewed.

Research paper thumbnail of Effect of Formaldehyde Inactivation on Poliovirus

Journal of Virology, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are pr... more Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

Research paper thumbnail of Synthesis of Immunogenic, but Noninfectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector

Journal of General Virology, 1989

SUMMARY A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear poly... more SUMMARY A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected

Research paper thumbnail of Immunogenicity and Antigenicity of Chimeric Picornaviruses Which Express Hepatitis A virus (HAV) peptide sequences: Evidence for a neutralization domain near the amino terminus of VP1 of HAV

Virology, 1992

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptid... more We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.

Research paper thumbnail of Antigenic and Molecular Evolution of the Vaccine Strain of Type 3 Poliovirus during the Period of Excretion by a Primary Vaccinee

Journal of General Virology, 1986

A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine str... more A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine strains of all three serotypes of poliovirus. The antigenic and molecular evolution of the Sabin strain of poliovirus type 3 was then followed throughout the entire period of virus excretion. Novel strains appeared at 8, 42 and 52 days post-vaccination and were the products of both intertypic recombination between type 2 and type 3 poliovirus in regions of the genome coding for non-structural proteins and of point mutations in the region coding for the structural proteins. Excretion of virus continued for 73 days. All strains examined reacted with all monoclonal antibodies specific for the main immunodominant antigenic site of type 3 poliovirus, but variation was observed at other, immunorecessive sites. These findings have possible implications for the evolution of the virus in vaccinees or in epidemics and are consistent with the known antigenic stability of the virus.

Research paper thumbnail of Reference materials for the evaluation of pre-mortem variant Creutzfeldt?Jakob disease diagnostic assays

Vox Sanguinis, 2007

A standard panel of materials is needed for the evaluation of assays being developed for the diag... more A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease. Tissues from human and animals incubating transmissible spongiform encephalopathy disease have been prepared, aliquoted and where possible characterized by in vitro methods. A standardized preparation of materials has been generated. Large-scale preparations of tissues and blood fractions can be used to directly compare the sensitivities of assays using different formats.

Research paper thumbnail of Working Standards for Hepatitis B Surface Antigen for Use in the UK Blood Transfusion Service: Results of a Collaborative Study

Vox Sanguinis, 1993

ABSTRACT

Research paper thumbnail of Testing Plasma Pools for Markers of Viral Contamination: The UK Experience

Vox Sanguinis, 1996

The transmission of viral infections through the use of products derived from blood has emphasise... more The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.

Research paper thumbnail of Human coronavirus 229E encodes a single ORF4 protein between the spike and the envelope genes

Virology journal, 2006

The genome of coronaviruses contains structural and non-structural genes, including several so-ca... more The genome of coronaviruses contains structural and non-structural genes, including several so-called accessory genes. All group 1b coronaviruses encode a single accessory protein between the spike and envelope genes, except for human coronavirus (HCoV) 229E. The prototype virus has a split gene, encoding the putative ORF4a and ORF4b proteins. To determine whether primary HCoV-229E isolates exhibit this unusual genome organization, we analyzed the ORF4a/b region of five current clinical isolates from The Netherlands and three early isolates collected at the Common Cold Unit (CCU) in Salisbury, UK. All Dutch isolates were identical in the ORF4a/b region at amino acid level. All CCU isolates are only 98% identical to the Dutch isolates at the nucleotide level, but more closely related to the prototype HCoV-229E (>98%). Remarkably, our analyses revealed that the laboratory adapted, prototype HCoV-229E has a 2-nucleotide deletion in the ORF4a/b region, whereas all clinical isolates c...

Research paper thumbnail of Characterization of the echovirus 7 receptor: domains of CD55 critical for virus binding

Journal of virology, 1995

CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates compleme... more CD55, or decay-accelerating factor (DAF), is a cell surface glycoprotein which regulates complement activity by accelerating the decay of C3/C5 convertases. Recently, we and others have established that this molecule acts as a cellular receptor for echovirus 7 and related viruses. DAF consists of five domains: four short consensus repeats (SCRs) and a serine/threonine-rich region, attached to the cell surface by a glycosylphosphatidyl inositol anchor. Chinese hamster ovary cells stably transfected with deletion mutants of DAF or DAF-membrane cofactor protein recombinants were analyzed for virus binding. The results indicate that the binding of echovirus 7 to DAF specifically requires SCR2, SCR3, and SCR4. There is also a nonspecific requirement for the S/T-rich region which probably functions to project the binding region away from the cell membrane. The three nonpeptide modifications of DAF, N-linked glycosylation, O-linked glycosylation, and the glycosylphosphatidyl inositol ancho...

Research paper thumbnail of Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus

Journal of virology, 1995

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from i... more Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensit...

Research paper thumbnail of Nucleic acid sequence of the region of the genome encoding capsid protein VP1 of neurovirulent and attenuated type 3 polioviruses

European journal of biochemistry / FEBS, Jan 3, 1983

Three closely related strains of poliovirus type 3 have been used to study the molecular basis of... more Three closely related strains of poliovirus type 3 have been used to study the molecular basis of attenuation in the currently used Sabin vaccine of this serotype. Plaque-purified derivatives of these strains possess closely similar serological and biochemical properties yet differ markedly in neurovirulence for monkeys. Molecular cloning via an RNA . cDNA method has facilitated comparative nucleotide sequencing. Initial efforts have concentrated on the region of the genome encoding VP1. Only minor structural differences between neurovirulent and attenuated type 3 strains were detected, in contrast to the major differences observed between the vaccine strains of poliovirus type 1 and its virulent precursor P1/Mahoney. These observations suggest that the molecular basis of attenuation of type 3 Sabin vaccine virus does not involve the VP1 polypeptide and, therefore, that mutations conferring the attenuated phenotype probably lie elsewhere in the genome.

Research paper thumbnail of Intertypic genomic rearrangements of poliovirus strains in vaccinees

Virology, 1988

In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus i... more In vivo intertypic RNA recombination has previously been observed in excreted type 3 poliovirus isolates from a normal asymptomatic primary vaccinee. This study examines isolates from additional primary vaccinees to determine whether intertypic recombination is a general occurrence in excreted polioviruses. T1 RNase oligonucleotide finger-printing and limited dideoxy primer extension RNA sequencing demonstrated no evidence of intertypic recombination among type 1 or type 2 excreted strains. However, vaccinees excreting type 3 strains for long periods of time eventually produced recombinant strains involving either type 1 or type 2 poliovirus. Moreover, a characteristic time course of appearance of excreted type 3 intertypic recombinant polioviruses was established. Type 3/type 1 and type 3/type 2 recombinant strains appeared at Days 10-11 with a single crossover site in the gene for nonstructural protein 2C. Type 3/type 2/type 3 complex recombinant strains with an additional crossov...

Research paper thumbnail of The 5' noncoding region of the type 2 poliovirus vaccine strain contains determinants of attenuation and temperature sensitivity

Virology, 1991

Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been gene... more Intratypic recombinants of P2/Sabin and P2/117, a neurovirulent vaccine revertant, have been generated in vitro using infectious cDNA clones and used to demonstrate that strong determinants of the attenuation and temperature-sensitive phenotypes of P2/Sabin reside in the 5' 492 nucleotides. In this region of the genome the viruses differ only at nucleotides 437 and 481. The ts phenotype associated with the 5' noncoding region is expressed at different temperatures in different cell lines, suggesting an involvement of cellular factors which may be species specific. Suppression of both the ts and attenuation phenotypes correlates with an A-G mutation at nucleotide 481, although other changes are also involved.

Research paper thumbnail of The Complete Nucleotide Sequence of Coxsackievirus B4 and Its Comparison to Other Members of the Picornaviridae

Journal of General Virology, 1987

SUMMARY The genome of the prototype strain of coxsackievirus B4 (J.V.B. Benschoten) has been clon... more SUMMARY The genome of the prototype strain of coxsackievirus B4 (J.V.B. Benschoten) has been cloned in Escherichia coil and its complete nucleotide sequence determined. Excluding the poly(A) tract, the RNA genome is 7395 nucleotides in length and appears to encode a single polyprotein of 2183 amino acids. The predicted amino acid sequence of the polyprotein shows close homology (88~) to

Research paper thumbnail of Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

Research paper thumbnail of No confirmation of pestivirus RNA in human virus vaccines

Journal of Clinical Microbiology

Recently, Harasawa and Tomiyama (1) reported the detection of pestivirus RNA in human vaccines. T... more Recently, Harasawa and Tomiyama (1) reported the detection of pestivirus RNA in human vaccines. The origin of the contamination was unknown, but the vaccines had been prepared in cell cultures supplemented with bovine fetal calf serum (FCS), which can be contaminated with bovine viral diarrhea virus (BVDV).

Research paper thumbnail of Comparing viral metagenomics methods using a highly multiplexed human viral pathogens reagent

Journal of Virological Methods, 2015

Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultan... more Unbiased metagenomic sequencing holds significant potential as a diagnostic tool for the simultaneous detection of any previously genetically described viral nucleic acids in clinical samples. Viral genome sequences can also inform on likely phenotypes including drug susceptibility or neutralization serotypes. In this study, different variables of the laboratory methods often used to generate viral metagenomics libraries were compared for their abilities to detect multiple viruses and generate full genome coverage. A biological reagent consisting of 25 different human RNA and DNA viral pathogens was used to estimate the effect of filtration and nuclease digestion, DNA/RNA extraction methods, pre-amplification and the use of different library preparation kits on the detection of viral nucleic acids. Filtration and nuclease treatment led to slight decreases in the percentage of viral sequence reads and number of viruses detected. For nucleic acid extractions silica spin columns improved viral sequence recovery relative to magnetic beads and Trizol extraction. Pre-amplification using random RT-PCR while generating more viral sequence reads resulted in detection of fewer viruses, more overlapping sequences, and lower genome coverage. The ScriptSeq library preparation method retrieved more viruses and a greater fraction of their genomes than the TruSeq and Nextera methods. Viral metagenomics sequencing was able to simultaneously detect up to 22 different viruses in the biological reagent analyzed including all those detected by qPCR. Further optimization will be required for the detection of viruses in biologically more complex samples such as tissues, blood, or feces.

Research paper thumbnail of Antigenic and Molecular Properties of Type 3 Poliovirus Responsible for an Outbreak of Poliomyelitis in a Vaccinated Population

Journal of General Virology, 1986

SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serological... more SUMMARY Virus isolated from an outbreak of poliomyelitis in Finland has been examined serologically and at the molecular level. The causative agent was an antigenically unusual strain of type 3 poliovirus, which was unrelated to the strains used to manufacture either live or killed poliovaccines. It is likely that the antigenic properties of the virus played a part in establishing

Research paper thumbnail of Isolation of SV40 from the environment of a colony of cynomolgus monkeys naturally infected with the virus

Virology, 2004

The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected ... more The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected with this virus has been analyzed by testing waste of the cage samples.

Research paper thumbnail of Current challenges in implementing cell-derived influenza vaccines: implications for production and regulation, July 2007, NIBSC, Potters Bar, UK

Vaccine, Jan 14, 2009

A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use o... more A meeting was held at NIBSC, UK in July 2007 to discuss the implications of progress in the use of cell culture systems for the manufacture of vaccines against influenza. Issues discussed included the effect of using eggs and different cell types in strain selection, development of seed viruses to be used in production and the nature of the reagents to be used in determining vaccine potency. Future studies to progress the field were reviewed.

Research paper thumbnail of Effect of Formaldehyde Inactivation on Poliovirus

Journal of Virology, 2014

Inactivated polio vaccines, which have been used in many countries for more than 50 years, are pr... more Inactivated polio vaccines, which have been used in many countries for more than 50 years, are produced by treating live poliovirus (PV) with formaldehyde. However, the molecular mechanisms underlying virus inactivation are not well understood. Infection by PV is initiated by virus binding to specific cell receptors, which results in viral particles undergoing sequential conformational changes that generate altered structural forms (135S and 80S particles) and leads to virus cell entry. We have analyzed the ability of inactivated PV to bind to the human poliovirus receptor (hPVR) using various techniques such as ultracentrifugation, fluorescence-activated cell sorting flow cytometry and real-time reverse transcription-PCR (RT-PCR). The results showed that although retaining the ability to bind to hPVR, inactivated PV bound less efficiently in comparison to live PV. We also found that inactivated PV showed resistance to structural conversion in vitro, as judged by measuring changes in antigenicity, the ability to bind to hPVR, and viral RNA release at high temperature. Furthermore, viral RNA from inactivated PV was shown to be modified, since cDNA yields obtained by RT-PCR amplification were severely reduced and no infectious virus was recovered after RNA transfection into susceptible cells.

Research paper thumbnail of Synthesis of Immunogenic, but Noninfectious, Poliovirus Particles in Insect Cells by a Baculovirus Expression Vector

Journal of General Virology, 1989

SUMMARY A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear poly... more SUMMARY A baculovirus expression vector (AcLeon) derived from Autographa californica nuclear polyhedrosis virus (AcNPV) was prepared containing the complete 6.6 kb coding region of the P3/Leon/37 strain of poliovirus type 3 placed under the control of the AcNPV polyhedrin promoter. The recombinant virus was used to infect Spodoptera frugiperda insect cells. As demonstrated by use of the appropriate antibodies, infected

Research paper thumbnail of Immunogenicity and Antigenicity of Chimeric Picornaviruses Which Express Hepatitis A virus (HAV) peptide sequences: Evidence for a neutralization domain near the amino terminus of VP1 of HAV

Virology, 1992

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptid... more We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera. Two chimeras (H15 and H2) stimulated production of low levels of HAV neutralizing antibodies in immunized rabbits or mice, although in both cases only a small fraction of immunized animals produced this response. The H15 chimera, which contains residues 13-24 of HAV VP1, elicited HAV neutralizing antibodies in three of nine rabbits and at least one of seven immunized mice. These results indicate that a neutralization domain exists in this region of VP1. However, human sera with high titers of antibodies to HAV failed to neutralize or immunoprecipitate this chimera, suggesting the absence of a significant antibody response to this neutralization domain following natural infection. Sera from rabbits immunized with H15 that did not develop HAV neutralizing antibodies contained antibodies reactive with the HAV peptide segment expressed by the H15 virus, indicating substantial differences in the specificities of antibodies elicited by this peptide segment among individual immunized rabbits. The H15 peptide insert was an effective antigen, as indicated by a high level of sensitivity of the H15 chimera to neutralization by a related anti-peptide antibody which was itself devoid of HAV neutralizing activity. One of 16 rabbits immunized with the H2 chimera (residues 101-108 of HAV VP1) developed HAV neutralizing antibodies, confirming both the presence and the highly conformational nature of a neutralization antigenic site involving these residues of HAV.

Research paper thumbnail of Antigenic and Molecular Evolution of the Vaccine Strain of Type 3 Poliovirus during the Period of Excretion by a Primary Vaccinee

Journal of General Virology, 1986

A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine str... more A 4 month old child was immunized with a vaccine containing the Sabin live attenuated vaccine strains of all three serotypes of poliovirus. The antigenic and molecular evolution of the Sabin strain of poliovirus type 3 was then followed throughout the entire period of virus excretion. Novel strains appeared at 8, 42 and 52 days post-vaccination and were the products of both intertypic recombination between type 2 and type 3 poliovirus in regions of the genome coding for non-structural proteins and of point mutations in the region coding for the structural proteins. Excretion of virus continued for 73 days. All strains examined reacted with all monoclonal antibodies specific for the main immunodominant antigenic site of type 3 poliovirus, but variation was observed at other, immunorecessive sites. These findings have possible implications for the evolution of the virus in vaccinees or in epidemics and are consistent with the known antigenic stability of the virus.

Research paper thumbnail of Reference materials for the evaluation of pre-mortem variant Creutzfeldt?Jakob disease diagnostic assays

Vox Sanguinis, 2007

A standard panel of materials is needed for the evaluation of assays being developed for the diag... more A standard panel of materials is needed for the evaluation of assays being developed for the diagnosis of variant Creutzfeldt-Jakob disease. Tissues from human and animals incubating transmissible spongiform encephalopathy disease have been prepared, aliquoted and where possible characterized by in vitro methods. A standardized preparation of materials has been generated. Large-scale preparations of tissues and blood fractions can be used to directly compare the sensitivities of assays using different formats.

Research paper thumbnail of Working Standards for Hepatitis B Surface Antigen for Use in the UK Blood Transfusion Service: Results of a Collaborative Study

Vox Sanguinis, 1993

ABSTRACT

Research paper thumbnail of Testing Plasma Pools for Markers of Viral Contamination: The UK Experience

Vox Sanguinis, 1996

The transmission of viral infections through the use of products derived from blood has emphasise... more The transmission of viral infections through the use of products derived from blood has emphasised the need for adequate validation of the production process, testing of materials used in production and quality control tests on the final product. Since the late 1980s, as part of its batch release procedures, NIBSC has tested for markers of viral infectivity plasma pools used in production of blood products used in the UK. As a result of testing over 9,000 pools, NIBSC has identified 9 pools contaminated with HBsAg and 2 pools containing antibodies to HIV-1. Since routine screening of plasma pools for anti-HCV was introduced in 1993, 8 pools out of the 4,000 tested have been found to contain antibodies to HCV. In addition, the release of 12 batches of blood products was withheld and it is known that further batches of material produced from the positive pools were not submitted for batch release. Studies involving assays of dilutions of known positive plasma samples indicated that there is considerable variation in the endpoint dilutions of antigen or antibody detected by test kits from different manufactures. The selection and validation of the kits used in such testing is therefore important. The usefulness of standardised low-level external controls in assays of plasma pools for markers of viral infection is discussed.