Glycoprotein G deletion mutants of equine herpesvirus 1 (EHV1; equine abortion virus) and EHV4 (equine rhinopneumonitis virus).

Glycoprotein G (gG) deletion mutants of EHV1 and EHV4, designated EHV1DeltagG and EHV4DeltagG, were constructed. The growth characteristics of the EHV1DeltagG mutants were similar to the parent virus. All of the EHV4DeltagG mutants grew more slowly in cell culture and produced plaques of different morphology including smaller size. The yields of both gG deletion mutant viruses in cell culture were similar to the parent viruses. Sequencing of the genes flanking gG, Southern blot, PCR and western blot analyses of the mutant viruses demonstrated that the deletions were as expected, except for EHV4DeltagG mutants, which in addition to deletion of gG contained unexpected deletions in the adjacent down stream gene ORF 71 (glycoprotein 2). Antisera to EHV1DeltagG and EHV4DeltagG neutralised the respective mutant and the parent viruses to the same titre and these antisera could be distinguished from antisera to the wild type viruses in a gG antibody detection ELISA. The mutant viruses may be useful as vaccine candidates and the deletion of gG may act as a marker to distinguish vaccinated from the naturally infected horses.

Puromycin-N-acetyltransferase as a selectable marker for use in Plasmodium falciparum.

The limited number of selectable markers available for malaria transfection has hindered extensive manipulation of the Plasmodium falciparum genome and subsequently thorough genetic analysis of this organism. In this paper, we demonstrate that P. falciparum is highly sensitive to the drug puromycin, but that transgenic expression of the puromycin-N-acetyltransferase (PAC) gene from Streptomyces alboninger confers resistance to this drug with the IC(50) and IC(90) values increasing approximately 3- and 7-fold, respectively in PAC-expressing parasites. Despite this relatively low level of resistance, parasite populations transfected with the PAC selectable marker and selected directly on puromycin emerged at the same rate post-transfection as human dihydrofolate reductase (hDHFR)-expressing parasites, selected independently with the anti-folate drug WR99210. Transfected parasites generally maintained the PAC expression plasmid episomally at between two and six copies per parasite. We also demonstrate by cycling transfected parasites in the presence and absence of puromycin for several weeks, that the PAC selectable marker can be used for gene-targeting. Since the mode of action of puromycin is distinct from other drugs currently used for the stable transfection of P. falciparum, the PAC selectable marker should also have applicability for use in conjunction with other positive selectable markers, thereby increasing the possibilities for more complex functional studies of this organism.

Evidence that Equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding.

Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as an Aphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathione S-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.

Antibodies against merozoite surface protein (MSP)-1(19) are a major component of the invasion-inhibitory response in individuals immune to malaria.

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.

An alteration in concatameric structure is associated with efficient segregation of plasmids in transfected Plasmodium falciparum parasites.

Transfection of the human malaria parasite Plasmodium falciparum is currently performed with circularised plasmids that are maintained episomally in parasites under drug selection but which are rapidly lost when selection pressure is removed. In this paper, we show that in instances where gene targeting is not favoured, transfected plasmids can change to stably replicating forms (SRFs) that are maintained episomally in the absence of drug selection. SRF DNA is a large concatamer of the parental plasmid comprising at least nine plasmids arranged in a head-to-tail array. We show as well that the original unstable replicating forms (URFs) are also present as head-to-tail concatamers, but only comprise three plasmids. Limited digestion and gamma irradiation experiments revealed that while URF concatamers are primarily circular, as expected, SRF concatamers form a more complex structure that includes extensive single-stranded DNA. No evidence of sequence rearrangement or additional sequence was detected in SRF DNA, including in transient replication experiments designed to select for more efficiently replicating plasmids. Surprisingly, these experiments revealed that the bacterial plasmid alone can replicate in parasites. Together, these results imply that transfected plasmids are required to form head-to-tail concatamers to be maintained in parasites and implicate both rolling-circle and recombination-dependent mechanisms in their replication.

Apical membrane antigen 1 plays a central role in erythrocyte invasion by Plasmodium species.

Apical membrane antigen 1 (AMA1) is an asexual blood-stage protein expressed in the invasive merozoite form of Plasmodia species, which are the causative agent of malaria. We have complemented the function of Plasmodium falciparum AMA1 (PfAMA1) with a divergent AMA1 transgene from Plasmodium chabaudi (PcAMA1). It was not possible to disrupt the PfAMA1 gene using 'knock-out' plasmids, although we demonstrate that the PfAMA1 gene can be targeted by homologous recombination. These experiments suggest that PfAMA1 is critical, perhaps essential, for blood-stage growth. Importantly, we showed that PcAMA1 expression in P. falciparum provides trans-species complementation to at least 35% of the function of endogenous PfAMA1 in human red cells. Furthermore, expression of this transgene in P. falciparum leads to more efficient invasion of murine erythrocytes. These results indicate an important role for AMA1 in the invasion of red blood cells (RBCs) across divergent Plasmodium species.

Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus.

Equine rhinitis A virus (ERAV) has recently been classified as an aphthovirus, a genus otherwise comprised of the different serotypes of Foot-and-mouth disease virus (FMDV). FMDV initiates translation via a type II internal ribosomal entry site (IRES) and utilizes two in-frame AUG codons to produce the leader proteinases Lab and Lb. Here we show that the ERAV 5' nontranslated region also possesses the core structures of a type II IRES. The functional activity of this region was characterized by transfection of bicistronic plasmids into BHK-21 cells. In this system the core type II structures, stem-loops D to L, in addition to a stem-loop (termed M) downstream of the first putative initiation codon, are required for translation of the second reporter gene. In FMDV, translation of Lb is more efficient than that of Lab despite the downstream location of the Lb AUG codon. The ERAV genome also has putative initiation sites in positions similar to those utilized in FMDV, except that in ERAV these are present as two AUG pairs (AUGAUG). Using the bicistronic expression system, we detected initiation from both AUG pairs, although in contrast to FMDV, the first site is strongly favored over the second. Mutational analysis of the AUG codons indicated that AUG2 is the major initiation site, although AUG1 can be accessed, albeit inefficiently, in the absence of AUG2. Further mutational analysis indicated that codons downstream of AUG2 appear to be accessed by a mechanism other than leaky scanning. Furthermore, we present preliminary evidence that it is possible for ribosomes to access downstream of the two AUG pairs. This study reveals important differences in IRES function between aphthoviruses.

Functional analysis of proteins involved in Plasmodium falciparum merozoite invasion of red blood cells.

Plasmodium falciparum causes the most lethal form of malaria in humans and is responsible for over two million deaths per year. The development of a vaccine against this parasite is an urgent priority and potential protein targets include those on the surface of the asexual merozoite stage, the form that invades the host erythrocyte. The development of methods to transfect P. falciparum has enabled the construction of gain-of-function and loss-of-function mutants and provided new strategies to analyse the role of parasite proteins. In this review, we describe the use of this technology to examine the role of merozoite antigens in erythrocyte invasion and to address their potential as vaccine candidates.

RAP1 controls rhoptry targeting of RAP2 in the malaria parasite Plasmodium falciparum.

Rhoptry associated protein 1 (RAP1) and 2 (RAP2), together with a poorly described third protein RAP3, form the low molecular weight complex within the rhoptries of Plasmodium falciparum. These proteins are thought to play a role in erythrocyte invasion by the extracellular merozoite and are important vaccine candidates. We used gene-targeting technology in P.falciparum blood-stage parasites to disrupt the RAP1 gene, producing parasites that express severely truncated forms of RAP1. Immunoprecipitation experiments suggest that truncated RAP1 species did not complex with RAP2 and RAP3. Consistent with this were the distinct subcellular localizations of RAP1 and 2 in disrupted RAP1 parasites, where RAP2 does not traffic to the rhoptries but is instead located in a compartment that appears related to the lumen of the endoplasmic reticulum. These results suggest that RAP1 is required to localize RAP2 to the rhoptries, supporting the hypothesis that rhoptry biogenesis is dependent in part on the secretory pathway in the parasite. The observation that apparently host-protective merozoite antigens are not essential for efficient erythrocyte invasion has important implications for vaccine design.

Targeted disruption of an erythrocyte binding antigen in Plasmodium falciparum is associated with a switch toward a sialic acid-independent pathway of invasion.

Erythrocyte invasion by Plasmodium requires molecules present both on the merozoite surface and within the specialized organelles of the apical complex. The Plasmodium erythrocyte binding protein family includes the Plasmodium falciparum sialic acid-binding protein, EBA-175 (erythrocyte binding antigen-175), which binds sialic acid present on glycophorin A of human erythrocytes. We address the role of the conserved 3'-cysteine rich region, the transmembrane, and cytoplasmic domains through targeted gene disruption. Truncation of EBA-175 had no measurable effect on either the level of EBA-175 protein expression or its subcellular localization. Similarly, there appears to be no impairment in the ability of soluble EBA-175 to be released into the culture supernatant after schizont rupture. Additionally, the 3'-cys rich region, transmembrane, and cytoplasmic domains of EBA-175 are apparently non-essential for merozoite invasion. In contrast, erythrocyte invasion via the EBA-175/glycophorin A route appears to have been disrupted to such a degree that the mutant lines have undergone a stable switch in invasion phenotype. As such, EBA-175 appears to have been functionally inactivated within the truncation mutants. The sialic acid-independent invasion pathway within the mutant parasites accounts for approximately 85% of invasion into normal erythrocytes. These data demonstrate the ability of P. falciparum to utilize alternate pathways for invasion of red blood cells, a property that most likely provides a substantial survival advantage in terms of overcoming host receptor heterogeneity and/or immune pressure.

Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid.

The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

Functional conservation of the malaria vaccine antigen MSP-119across distantly related Plasmodium species.

The C-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP-119) is at present a leading malaria vaccine candidate. Antibodies against the epidermal growth factor-like domains of MSP-1 19are associated with immunity to P. falciparum and active immunization with recombinant forms of the molecule protect against malaria challenge in various experimental systems. These findings, with the knowledge that epidermal growth factor-like domains in other molecules have essential binding functions, indicate the importance of this protein in merozoite invasion of red blood cells. Despite extensive molecular epidemiological investigations, only limited sequence polymorphism has been identified in P. falciparum MSP-119 (refs. 9-11). This indicates its sequence is functionally constrained, and is used in support of the use of MSP-119 as a vaccine. Here, we have successfully complemented the function of most of P. falciparum MSP-119 with the corresponding but highly divergent sequence from the rodent parasite P. chabaudi. The results indicate that the role of MSP-119 in red blood cell invasion is conserved across distantly related Plasmodium species and show that the sequence of P. falciparum MSP-119 is not constrained by function.

Transfection of the human malaria parasite Plasmodium falciparum.

In the past few years, methods have been developed which allow the introduction of exogenous DNA into the human malaria parasite Plasmodium falciparum. This important technical advance known as parasite transfection, provides powerful new tools to study the function of Plasmodium proteins and their roles in biology and disease. Already it has allowed the analysis of promoter function and has been successfully applied to establish the role of particular molecules and/or mutations in the biology of this parasite. This review summarises the current state of the technology and how it has been applied to dissect the function of the P. falciparum genome.

Equine herpesvirus-4 glycoprotein G is secreted as a disulphide-linked homodimer and is present as two homodimeric species in the virion.

Glycoprotein G (gG) homologues have been found in most alphaherpesviruses although little is known about their structure or function. In this study, three species of equine herpesvirus-4 (EHV-4) gG were identified: a full-length 68 kDa virion-associated species (gGVL), a 12 kDa virion-associated species (gGVS) and a 60 kDa secreted species (gGS), detected in the medium of infected cells. gGS and gGVS appear to be proteolytic cleavage products of gGVL and correspond to the N- and C-terminal regions, respectively. It was shown that gGS and gGVL are similarly glycosylated possessing mostly N-linked complex-type carbohydrate side chains. Western blots of proteins separated under nonreducing conditions established that gGS is secreted as a 120 kDa glycoprotein while the virion-associated species, gGVL and gGVS, are present in the virion as 140 and 20 kDa proteins, respectively. As gGS and gGVL do not appear to associate stably with other viral proteins, it is most likely that each species exists as a disulphide-linked homodimer. Pulse-chase experiments indicated that gGVL is rapidly assembled as a homodimer prior to both carbohydrate side-chain maturation in the Golgi and proteolytic cleavage. Proteolytic cleavage of full-length gG occurs during or immediately after passage through the Golgi. Secreted and virion-associated species of gG were identified in the closely related virus EHV-1 and were of similar molecular masses to the corresponding EHV-4 gG species.

Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses.

Equine rhinovirus 1 (ERhV1) is a recognized cause of acute febrile respiratory disease in horse, although the virus is rarely isolated from such animals, despite seroprevalence rates as high as 50% in some horse populations. Recently, ERhV1 has been shown to be most closely related to foot-and-mouth disease virus, raising questions as to its disease associations in horses. We report that ERhV1 infection was the likely cause of two separate outbreaks of severe febrile respiratory disease which involved more than 20 horses. Attempts to isolate ErhV1 from nasopharyngeal swabs by conventional cell culture methods were unsuccessful, in that cytopathology was not observed. Viral antigen was detected by immunofluorescence assay in the cytoplasm of cells infected with 10 of 15 nasopharyngeal swab samples, indicating the presence and presumably replication of ERhV1. A rise in serum neutralizing antibody titer between acute- and convalescent-phase sera confirmed that ERhV1 was causatively associated with one of the outbreaks. ERhV1 RNA was detected in nasopharyngeal swabs collected from all horses during the acute phase of disease by reverse transcription-PCR. Nucleotide sequencing of amplified products showed that within each outbreak a single strain of ERhV1 was involved but that distinct viruses were involved in each outbreak. A retrospective study of samples from nine other outbreaks of respiratory disease in horses suggested ERhV1 etiology in at least two of these. We conclude that the relative importance of ERhV1 as a cause of acute febrile respiratory disease in horses has been underestimated due to failure in many instances to isolate virus by conventional cell culture methods.

Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress.

Knobs at the surface of erythrocytes infected with Plasmodium falciparum have been proposed to be important in adherence of these cells to the vascular endothelium. This structure contains the knob-associated histidine-rich protein (KAHRP) and the adhesion receptor P. falciparum erythrocyte membrane protein 1. We have disrupted the gene encoding KAHRP and show that it is essential for knob formation. Knob-transfectants adhere to CD36 in static assays; when tested under flow conditions that mimic those of postcapillary venules, however, the binding to CD36 was dramatically reduced. These data suggest that knobs on P. falciparum-infected erythrocytes exert an important influence on adherence of parasitized-erythrocytes to microvascular endothelium, an important process in the pathogenesis of P. falciparum infections.

Stable transgene expression in Plasmodium falciparum.

Plasmid vectors designed to express transgenes and a selectable marker in Plasmodiumfalciparum were constructed. These consist of a selectable gene cassette comprising the Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene mutated to confer pyrimethamine resistance flanked by either Plasmodium chabaudi DHFR-TS or P. falciparum calmodulin promoter sequences and the P. falciparum histidine rich protein 2 3' region. Also, each vector includes a different expression cassette driven by various Plasmodium transcriptional control sequences. Initially, the chloramphenicol acetyl transferase (CAT) reporter gene was cloned into the expression site of two vectors, pCC6-CAT and pCC13-CAT, which were identical except for the orientation of the expression cassette with respect to the selectable gene cassette. Approximately 8-fold more CAT activity was detected when the direction of transcription of the expression cassettes was in a head to head, rather than a tail to head, orientation. Importantly, it was found that stable transfection could only be achieved when the gene cassettes were in the head to head direction suggesting that this orientation also has an effect on the level of expression of the selectable marker. All other plasmids were designed with the cassettes in a head to head orientation. With the exception of pCC6-CAT and a second vector pHC4-CAT, stable transfectants were obtained with each vector in which the CAT gene had been inserted into the expression cassette. This is the first time vectors for the stable expression in Plasmodium parasites of transgenes other than a selectable marker have been described.

Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest.

Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses.

Equine rhinovirus 1 (ERhV1) is a respiratory pathogen of horses which has an uncertain taxonomic status. We have determined the nucleotide sequence of the ERhV1 genome except for a small region at the 5' end. The predicted polyprotein was encoded by 6741 nucleotides and possessed a typical picornavirus proteolytic cleavage pattern, including a leader polypeptide. The genomic structure and predicted amino acid sequence of ERhV1 were more similar to those of foot-and-mouth disease viruses (FMDVs), the only members of the aphthovirus genus, than to those of other picornaviruses. Features which were most similar to FMDV included a 16-amino acid 2A protein which was 87.5% identical in sequence of FMDV 2A, a leader (L) protein similar in size to FMDV Lab and the possibility of a truncated L protein similar in size to FMDV Lb, and a 3C protease which recognizes different cleavage sites. However, unlike FMDV, ERhV1 had only one copy of the 3B (VPg) polypeptide. The phylogenetic relationships of the ERhV1 sequence and nucleotide sequences of representative species of the five genera of the family Picornaviridae were examined. Nucleotide sequences coding for the complete polyprotein, the RNA polymerase, and VP1 were analyzed separately. The phylogenetic trees confirmed that ERhV1 was more closely related to FMDV than to other picornaviruses and suggested that ERhV1 may be a member, albeit very distant, of the aphthovirus genus.