Paul R. Lambden

Development of a Transformation System for Chlamydia trachomatis: Restoration of Glycogen Biosynthesis by Acquisition of a Plasmid Shuttle Vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.

Organization and Expression of Calicivirus Genes

The application of molecular techniques to the characterization of caliciviruses has resulted in an extensive database of sequence information. This information has led to the identification of 4 distinct genera. The human enteric caliciviruses have been assigned to 2 of these genera. This division is reflected not only in sequence diversity but in a fundamental difference in genome organization. Complete genome sequences are now available for 5 enteric caliciviruses and demonstrate that human and animal enteric caliciviruses are phylogenetically closely related. Currently, there is no cell culture system for the human viruses; therefore, studies have relied on heterologous expression and in vitro systems. These studies have shown that in both human and animal viruses the viral nonstructural proteins are produced from a polyprotein precursor that is cleaved by a single viral protease. The purpose of this article is to provide an overview of the current knowledge of genome structure and gene expression in the enteric caliciviruses.

Point mutation in meningococcal por A gene associated with increased endemic disease

The por A gene, which encodes expression of meningococcal class 1 outer membrane protein, responsible for antigenic subtype specificity, has been cloned and sequenced in an isolate of Neisseria meningitidis (B:15:P1.7,16) from a patient in the Gloucester area with meningococcal meningitis. Comparison of the sequence with that of the equivalent gene from the P1.7,16 reference strain reveals a point mutation which generates a single aminoacid change in the epitope responsible for P1.16 specificity. Monoclonal antibodies with P1.16 specificity do not react with synthetic peptides that correspond to the altered epitope, and do not promote complement-mediated bactericidal killing of the isolate. Analysis of other strains shows widespread distribution of infections due to B:15:P1.7,16 meningococci with the altered epitope (P1.16b) in England and Wales.

Variations in Surface Protein Composition Associated with Virulence Properties in Opacity Types of Neisseria gonorrhoeae

The biological properties of a series of opacity variants of Neisseria gonorrhoeae P9 have been examined. A novel protein, designated protein IId* (mol. wt 28 850), was identified within the set of heat-modifiable surface proteins previously reported. All variants producing extra outer membrane proteins were less sensitive to the bactericidal action of serum than the prototype transparent strain, with protein IIa* (mol. wt 28 500) being associated with increased resistance. The production of a different protein, protein II* (mol. wt 29 000), was correlated with resistance to low molecular weight antimicrobial agents (penicillin, fusidic acid, Cu2+, Zn2+). Increased adhesion to human buccal epithelial cells was demonstrated in all variants that produced extra surface proteins. These variants did not show increased binding to hexyl- and phenyl-substituted Sepharose gels suggesting that hydrophobic interaction was not responsible for their cohesive properties. The prototype strain lacking additional proteins demonstrated the greatest binding to erythrocytes, indicating that adhesion to buccal cells and red blood cells is mediated by different mechanisms. One variant producing protein IIa* showed increased association with leukocytes, whereas another producing protein IIb* showed decreased association with leukocytes. These results show that the heat-modifiable surface proteins are important virulence attributes of the gonococcus: this must be considered in the selection of strains for vaccine trials.

Human enteric caliciviruses have a unique genome structure and are distinct from the Norwalk-like viruses

SummaryClassic human enteric caliciviruses (HuCVs) have a distinctive morphology and are primarily associated with pediatric acute gastroenteritis. Although morphologically distinct from the small round structured viruses (SRSVs), the classic HuCVs are thought to be closely related and were anticipated to have a similar genome organisation. We report the first genome sequence and molecular characterisation of a classic human enteric calicivirus associated with a case of acute vomiting and diarrhoea in an infant. The RNA genome (7266 nt) is smaller than the genome of SRSVs from the two genetic groups and has a unique arrangement of open reading frames. Further analysis of the 3′ terminal 3 kb from a second unrelated isolate confirmed this genomic organisation. Analysis of capsid and RNA polymerase sequences together with the unique genomic organisation of classic HuCV suggest these viruses are more closely related to the animal caliciviruses than the enteric SRSV group of viruses.

The molecular biology of caliciviruses

IP: 54.70.40.11 On: Thu, 25 Oct 2018 09:25:47 Journal of General Virology (1997), 78, 291–301. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Parkville virus : A novel genetic variant of human calicivirus in the Sapporo virus clade, associated with an outbreak of gastroenteritis in adults

This report describes the characterization of Parkville virus, the etiologic agent of an outbreak of foodborne gastroenteritis, that has the morphology of a calicivirus and genetic properties that distinguish it from previously identified strains in the Sapporo/Manchester virus clade. Sequence analysis of the Parkville virus genome showed it contained the RNA‐dependent RNA polymerase motifs GLPSG and YGDD characteristic of members of the family Caliciviridae with an organization identical to that reported for the Manchester virus where the capsid region of the polyprotein is fused to the RNA polymerase. Parkville virus however, demonstrates considerable sequence divergence from both the Manchester and Sapporo caliciviruses, providing the first indications that genetic diversity exists within caliciviruses of this previously homogeneous clade. On the basis of recent advances in the genetic characterization of members of the family Caliciviridae, we propose a new interim phylogenetic classification system in which Parkville virus would be included with Manchester and Sapporo virus as a separate group distinct from the small round‐structured viruses (Norwalk‐like viruses) that also cause diarrhea in humans. J. Med. Virol. 52:173–178, 1997.

Class 1 outer membrane protein of Neisseria meningitidis: epitope analysis of the antigenic diversity between strains, implications for subtype definition and molecular epidemiology.

The VR1 and VR2 regions of the class 1 protein have been sequenced from a number of meningococcal strains, including non‐subtypable strains and strains of apparently identical subtype. The amino acid sequences have been used to construct synthetic peptides for mapping subtype‐specific epitopes. The majority of epitopes was found to be located in VR2 at the apex of a predicted surface‐exposed loop. A single amino acid change within an epitope, or an amino acid deletion outside an epitope, were both associated with loss of subtype specificity, resulting from a change in the predicted conformation at the apex of the loop structure. Analysis of the sequence information combined with knowledge of defined epitopes also revealed considerable additional information not demonstrated by current subtyping procedures.

Antibodies to Respiratory Syncytial Virus Polypeptides and their Significance in Human Infection

The human antibody response to respiratory syncytial (RS) virus infection was investigated using radioimmunoprecipitation analysis (RIPA). A total of nine RS virus-specific proteins, VP200, VGP95, VP68, VGP48, VPN41, VP35, VP27, VP23 and VGP20 were identified by comparing 35S- or 3H-labelled extracts of infected and uninfected HEp-2 cells, and by radioimmunoprecipitation using a hyperimmune human serum. Three glycopeptides, VGP95, VGP48 and VGP20, were identified by incorporation of [3H]glucosamine, and two of these (VGP48 and VGP20) were assumed to be part of a single disulphide-bonded polypeptide since they were precipitated by a monoclonal antibody raised against a surface protein. Human serum antibodies to three major RS virus proteins, VGP95, VGP48/VGP20 and VPN41 were measured by RIPA using radioiodinated RS virus antigens. Sera from a group of mothers whose babies escaped RS virus infection during a local epidemic showed increased antibody levels to VPN41 when compared to sera from mothers whose babies had become infected with RS virus within the first 6 months of life. In infants who remained uninfected with RS virus during the first 12 months of life the maternal gift of antibody decayed to about 50% at 3 months with traces of antibodies detected in a few sera at 12 months. The antibody levels detected in the sera of infants less than 3 months old convalescent from primary RS virus infection did not exceed the mean levels present in the serum of uninfected babies. Infants between the ages of 6 and 12 months were able to mount an IgG response to VPN41 and VGP48 but, unlike adults and older children, a particularly striking finding was their failure to produce antibodies to VGP95.

Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA

Noroviruses are the major cause of nonbacterial gastroenteritis in humans. These viruses have remained refractory to detailed molecular studies because of the lack of a reverse genetics system coupled to a permissive cell line for targeted genetic manipulation. There is no permissive cell line in which to grow infectious human noroviruses nor an authentic animal model that supports their replication. In contrast, murine norovirus (MNV) offers a tractable system for the study of noroviruses with the recent discovery of permissive cells and a mouse model. The lack of a reverse genetic system for MNV has been a significant block to understanding the biology of noroviruses. We report recovery of infectious MNV after baculovirus delivery of viral cDNA to human hepatoma cells under the control of an inducible DNA polymerase (pol) II promoter. Recovered virus replicated in murine macrophage (RAW264.7) cells, and the recovery of MNV from DNA was confirmed through recovery of virus containing a marker mutation. This pol II promoter driven expression of viral cDNA also generated infectious virus after transfection of HEK293T cells, thus providing both transduction and transfection systems for norovirus reverse genetics. We used norovirus reverse genetics to demonstrate by mutagenesis of the protease–polymerase (pro–pol) cleavage site that processing of pro–pol is essential for the recovery of infectious MNV. This represents the first infectious reverse genetics system for a norovirus, and should provide approaches to address fundamental questions in norovirus molecular biology and replication.