Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

University of Melbourne

The mechanisms of foot-and-mouth disease virus (FMDV) persistence are poorly understood. It is thought the existence of viral quasispecies that encompass sub-populations with varying survival competencies and antigenicities may play some role in the maintenance of virus in persistently infected animals. By analyzing nucleotide sequences encoding the viral VP2 protein in oesophageal-pharyngeal fluid (probang) samples from cattle at different stages of infection, the significance of any amino acid changes in relation to persistence was investigated. Twenty-two experimentally infected cattle (including six carriers) from three animal experiments with FMDV type O UKG34/2001 were studied. Comparison of VP2 sequences in these samples with the inoculum sequence revealed a consistent change in the B-C loop in FMDV from persistently infected cattle. Residue 2079 changed from Y to H in five carrier animals and residue 2080 changed from A to Q in one carrier from 14 days post-infection onward. In contrast, there were no changes evident in any of the non-carriers up to 28 days post-infection. The results indicate that a substitution change in the B-C loop of VP2 may be associated with persistent FMDV infection in cattle.

Consistent change in the B-C loop of VP2 observed in foot-and-mouth disease virus from persistently infected cattle: implications for association with persistence.

Foot-and-mouth disease virus (FMDV) is a positive-sense, single stranded RNA virus and its replication involves the synthesis of a negative strand intermediate. In the present study, a strand-specific quantitative RT-PCR assay was developed for analysis of FMDV replication. Strand-specific detection of viral positive and negative strand RNA was achieved using a high reverse transcription (RT) temperature (62 degrees C) and a tagged RT primer. In both the positive and negative strand assays, the lowest reliably detectable concentration was 1 x 10(2) copies/microl. The assays developed were successfully used to analyse viral replication in tissues collected from experimentally infected sheep during both acute and persistent infection. The results showed that while replication was observed in all tissues examined during acute infection, active viral replication during persistent infection was only detected in the tonsil. These results are consistent with the current opinion that the tonsil in sheep is the main predilection site for virus persistence. This assay will be used in the future to look further at replication in experimentally infected animals, including the study of individual cell types, and will improve our understanding of FMDV pathogenesis.

Analysis of foot-and-mouth disease virus replication using strand-specific quantitative RT-PCR.

Foot-and-mouth disease (FMD) in adult sheep usually causes milder clinical signs than in cattle or pigs, and is often subtle enough to go undiagnosed. In contrast, FMD in lambs has been reported to cause high mortality during field outbreaks. In order to investigate the pathogenesis of FMD in lambs, two groups, aged 10-14 days, were infected with foot-and-mouth disease virus (FMDV) type O UKG. One group of lambs (n=8) was inoculated with FMDV in the coronary band, while the other (n=4) was infected by direct contact with FMDV-inoculated ewes. Daily serum samples and temperature measurements were taken. Lambs were killed sequentially and tissue samples taken for analysis. Using real-time RT-PCR, viral RNA levels in tissue samples and serum were measured, and a novel strand-specific real-time RT-PCR assay was used to quantify viral replication levels in tissues. Tissue sections were examined for histopathological lesions, and in situ hybridisation (ISH) was used to localise viral RNA within histological sections. The contact-infected lambs became infected approximately 24h after the ewes were inoculated. Vesicular lesions developed on the feet of all lambs and on the caudo-lateral part of the tongues of six of the eight inoculated lambs and three of the four contact-infected lambs. Although no lambs developed severe clinical signs, one of the contact-infected lambs died acutely at 5 days post-exposure. Histological examination of the heart from this lamb showed multi-focal areas of lymphocytic-plasmacytic myocarditis; similar lesions were also observed in the hearts of three of the inoculated lambs. Using ISH, viral RNA was localised within cardiac and skeletal muscle cells from the lamb which had died, and also from vesicular lesions on the coronary band and tongue of inoculated lambs. These results provide a detailed description of the pathogenesis of the disease in lambs.

Foot-and-mouth disease virus infection in young lambs: pathogenesis and tissue tropism.

Equine rhinitis B virus (ERBV) is the single species in the genus Erbovirus, family Picornaviridae. Equine rhinitis B viruses exist in three serotypes and are associated with respiratory disease in horses. Members of the species vary in stability at acid pH. To date there has been discordance in genotype, serotype and acid stability phenotype groupings. To identify capsid regions associated with acid stability, two viruses were serially treated at pH 3.3 to isolate acid-stable mutants. An acid-stable mutant of the prototype acid-labile serotype 1 virus contained a single amino acid change in the C-terminus of VP1. Similar treatment with a separate isolate identified a multiple ERBV serotype infection with acid-labile serotype 1 and acid-stable serotype 3. Dual infections were subsequently identified in original swabs taken from the infected horse and from two further cell culture passaged viruses originally isolated in Switzerland. Serotype specific rat antisera were produced and used to examine a collection of isolates from a range of genotypes, acid stability phenotypes and serotypes. In contrast to previous reports, we showed viruses previously classified as acid-stable serotype 1 are in fact serotype 3 and that there is a clear association of serotype with genotype and acid stability phenotype in ERBVs. Additionally, we have shown that ERBV capsids dissociate into pentamers in acidic conditions below their threshold of stability, similarly to closely related viruses in the same family.

Identification of mixed equine rhinitis B virus infections leading to further insight on the relationship between genotype, serotype and acid stability phenotype.

Serology is used to predict vaccine induced protection against challenge with a heterologous strain of the same serotype of foot-and-mouth disease virus (FMDV). To evaluate the accuracy of such predictions, we compared the protection afforded to cattle vaccinated with the O(1) Manisa strain of FMDV against challenge with either a homologous (O(1) Manisa) or a heterologous strain (O(1) Campos). Serology by virus neutralization test (VNT) using O(1) Manisa antiserum predicted an acceptable protection against such a challenge. Two experiments were carried out to compare the results for consistency. A total of 78 naïve cattle were vaccinated with different antigen payloads (60-0.94 μg) of O(1) Manisa. They were challenged by intradermolingual inoculation with live FMDV, either O(1) Manisa or O(1) Campos. Unvaccinated naïve control cattle (n=20) were also challenged with either the O(1) Manisa or O(1) Campos viruses and all developed generalized FMD. The protection results for the vaccinated cattle revealed that higher payloads of O(1) Manisa vaccine were needed to protect against heterologous challenge compared to that for homologous challenge. The 50% protective dose (PD(50)) values for the vaccine in experiments 1 and 2 were found to be 28.78 and 9.44 for the homologous challenge and 3.98 and 5.01 for heterologous challenge. Furthermore, protection against O(1) Campos required a higher level of vaccine-induced antibody against this virus compared to the level of O(1) Manisa neutralizing antibody associated with protection against homologous challenge. The 50% protective level of in vitro neutralizing antibody was found to be log(10)1.827 for O(1) Campos and log(10)0.954 for O(1) Manisa based on O(1) Manisa based virus neutralization test.

Evaluation of cross-protection between O1 Manisa and O1 Campos in cattle vaccinated with foot-and-mouth disease virus vaccine incorporating different payloads of inactivated O1 Manisa antigen.

Erbovirus is a genus of the family Picornaviridae and equine rhinitis B virus (ERBV) is the sole species. Erboviruses infect horses causing acute respiratory disease and sub-clinical and persistent infections. Despite the high seroprevalence and worldwide distribution of these viruses, the pathogenesis and antigenic structure of the three ERBV serotypes (ERBV1, 2 and 3) is poorly understood. To characterise linear epitopes on ERBV structural proteins, a set of fusion proteins were expressed in Escherichia coli. These proteins were tested in Western blot and ELISA and reactive proteins were also used to identify neutralisation epitopes. VP1 contained serotype specific epitopes whereas VP2 was highly cross-reactive across the serotypes. The C-terminus of VP1 accounted for most of the reactivity of full-length VP1 and was also the location of a neutralising site in each serotype.

Mapping B-cell epitopes in equine rhinitis B viruses and identification of a neutralising site in the VP1 C-terminus.

Egress of wrapped virus (WV) to the cell periphery following vaccinia virus (VACV) replication is dependent on interactions with the microtubule motor complex kinesin-1 and is mediated by the viral envelope protein A36. Here we report that ectromelia virus (ECTV), a related orthopoxvirus and the causative agent of mousepox, encodes an A36 homologue (ECTV-Mos-142) that is highly conserved despite a large truncation at the C terminus. Deleting the ECTV A36R gene leads to a reduction in the number of extracellular viruses formed and to a reduced plaque size, consistent with a role in microtubule transport. We also observed a complete loss of virus-associated actin comets, another phenotype dependent on A36 expression during VACV infection. ECTV ΔA36R was severely attenuated when used to infect the normally susceptible BALB/c mouse strain. ECTV ΔA36R replication and spread from the draining lymph nodes to the liver and spleen were significantly reduced in BALB/c mice and in Rag-1-deficient mice, which lack T and B lymphocytes. The dramatic reduction in ECTV ΔA36R titers early during the course of infection was not associated with an augmented immune response. Taken together, these findings demonstrate the critical role that subcellular transport pathways play not only in orthopoxvirus infection in an in vitro context but also during orthopoxvirus pathogenesis in a natural host. Furthermore, despite the attenuation of the mutant virus, we found that infection nonetheless induced protective immunity in mice, suggesting that orthopoxvirus vectors with A36 deletions may be considered another safe vaccine alternative.

Loss of cytoskeletal transport during egress critically attenuates ectromelia virus infection in vivo.

The study of host-pathogen interactions over past decades has benefited from advances in microscopy and fluorescent imaging techniques. A particularly powerful model in this field is vaccinia virus (VACV), which due to its amenability to genetic manipulation has been a productive model in advancing the understanding of the transport of subcellular cargoes. Conventional light microscopy imposes an upper limit of resolution of ~250nm, hence knowledge of events occurring at the sub-viral resolution is based predominantly on studies utilising electron microscopy. The development of super-resolution light microscopy presents the opportunity to bridge the gap between these two technologies. This report describes the analysis of VACV replication using fluorescent recombinant viruses, achieving sub-viral resolution with three-dimensional structured illumination microscopy. This is the first report of successfully resolving poxvirus particle morphologies at the scale of single virus particles using light microscopy.

Sub-viral imaging of vaccinia virus using super-resolution microscopy.

Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36(YdF) virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36(YdF) infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36(YdF) extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5(P189S) mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.

A36-dependent actin filament nucleation promotes release of vaccinia virus.

Of the many members that comprise the family Picornaviridae, only two species are known to infect horses: equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV). Each species now occupies a distinct phylogenetic branch within the family, with the single serotype of ERAV grouping with the aphthoviruses, such as foot-and-mouth disease virus (FMDV), and the three serotypes of ERBV as the sole members of the genus Erbovirus. The high seroprevalence of equine picornaviruses in horse populations worldwide contrasts with the relatively few reports of detection of these viruses and poor understanding of their contribution to disease. This review examines the current knowledge regarding the distribution and pathogenesis of these viruses and discusses recent advances in diagnostic methods that may lead to a better understanding of the role of these viruses as contributors to equine respiratory disease.

Equine picornaviruses: Well known but poorly understood.

Respiratory infections are a major burden in the performance horse industry. Equine rhinitis B virus (ERBV) has been isolated from horses displaying clinical respiratory disease, and ERBV-neutralizing antibodies have been detected in 50-80% of horses in reported surveys. Current ERBV isolation and detection methods may underestimate the number of ERBV-positive animals and do not identify multiple serotype infections. The aim of the current study was to develop a serotyping ERBV antibody-detection enzyme-linked immunosorbent assay (ELISA) and examine the seroprevalence of ERBV in a group of Australian weanling horses. ELISAs with high sensitivity and specificity were developed. The seroprevalence of ERBV in the weanling horses was high (74-86%); ERBV-3 antibodies were most prevalent (58-62%) and ERBV-2 antibodies were least prevalent (10-16%). Many horses were seropositive to 2 or more serotypes. All 3 serotypes of ERBV were detected, and concurrent positivity to multiple serotypes was common.

Jacquelyn Horsington

Asia Pacific Centre for Animal Health,
Faculty of Veterinary Science,
Asia Pacific Centre for Animal Health, Faculty of Veterinary Science

University of Melbourne

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Jacquelyn Horsington

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University of Melbourne

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Asia Pacific Centre for Animal Health,
Faculty of Veterinary Science,
Asia Pacific Centre for Animal Health, Faculty of Veterinary Science