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Evidence Project Final Report

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 20

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NoteIn line with the Freedom of Information Act 2000, Defra aims to place the results of its completed research projects in the public domain wherever possible. The Evidence Project Final Report is designed to capture the information on the results and outputs of Defra-funded research in a format that is easily publishable through the Defra websiteAn Evidence Project Final Report must be completed for all projects.

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Project identification

1. Defra Project code SE0424

2. Project title

Genetic and antigenic characterisation of new and emerging lyssaviruses that pose a threat to the UK

3. Contractororganisation(s)

Animal Health and Veterinary Laboratories AgencyWoodham LaneNew HawAddlestoneSurrey KT15 3NB

54. Total Defra project costs £359,767(agreed fixed price)

5. Project: start date................ 01/04/2009

end date................. 30/09/2013


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so...................................................................................YES NO (a) When preparing Evidence Project Final Reports contractors should bear in mind that Defra intends that

they be made public. They should be written in a clear and concise manner and represent a full account of the research project which someone not closely associated with the project can follow.Defra recognises that in a small minority of cases there may be information, such as intellectual property or commercially confidential data, used in or generated by the research project, which should not be disclosed. In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain. Where it is impossible to complete the Final Report without including references to any sensitive or confidential data, the information should be included and section (b) completed. NB: only in exceptional circumstances will Defra expect contractors to give a "No" answer.In all cases, reasons for withholding information must be fully in line with exemptions under the Environmental Information Regulations or the Freedom of Information Act 2000.

(b) If you have answered NO, please explain why the Final report should not be released into public domain

Executive Summary7. The executive summary must not exceed 2 sides in total of A4 and should be understandable to the

intelligent non-scientist. It should cover the main objectives, methods and findings of the research, together with any other significant events and options for new work.

This project successfully employed three differing approaches to develop and apply tools to study the immune responses and potential to cause clinical disease for lyssaviruses, which may pose a threat to UK veterinary public health. In addition, we have obtained and published the genome sequence of the novel lyssavirus (Ikoma lyssavirus) identified in an African civet by APHA scientists. This finding further extended our knowledge of the potential diversity of global lyssavirus isolates. Whilst the observation of a new virus in a mammal in Africa may at first have little relevance to the EU, the detection of a further highly divergent lyssavirus, Lleida virus in European bats demonstrates that it is important that our capabilities cover detection of all lyssaviruses. To have confidence that tests could detect a virus in the context of human and veterinary health, we must be confident that our diagnostic portfolio can detect all known related viruses.

Objective 01 Application of Reverse Genetics Technology to study the role of the EBLV proteins in viral pathogenicity and neuroinvasiveness. [35 months]

Objective: In collaboration with Thomas Jefferson University, we will construct EBLV infectious clones in which the individual protein genes have been replaced by those from other lyssaviruses. This will facilitate a greater understanding of the role played by these individual proteins in viral pathogenicity, neuroinvasiveness and immune evasion.

European bat lyssaviruses type 1 (EBLV-1) and type 2 (EBLV-2) circulate within bat populations throughout Europe and are capable of causing disease indistinguishable from that caused by classical rabies virus (RABV). Lyssaviruses, including RABV, can vary significantly in their pathogenicity and host susceptibility. Whilst RABV variants demonstrate a world wide range of mammalian hosts, some other lyssavirus species appear to be restricted geographically and maintained in relatively few or single hosts. The determinants of host susceptibility and pathogenicity are poorly understood. Using reverse genetics technology, we have demonstrated the importance of the source of the lyssavirus glycoprotein with respect to virus growth in tissue culture and pathogenicity in a mouse model. We replaced the glycoprotein of a laboratory adapted non-pathogenic RABV with the glycoproteins of the pathogenic European bat lyssaviruses. The replacement of the glycoprotein rendered the recombinant viruses pathogenic in mice but to a lesser extent than the respective wild type EBLVs. Histopathological changes and antigen distribution observed for the EBLV recombinant viruses were more representative of those observed for the non-pathogenic virus than for wild type EBLV, suggesting that whilst inserting the glycoprotein of a pathogenic virus reduced survival other viral proteins play a greater role in virus replication and


dissemination. Using antigenic mapping techniques (Antigenic Cartography), we confirmed that the glycoprotein is the dominant antigenic component of lyssaviruses. These findings provide an excellent platform for the combined use of reverse genetics, in vivo pathogenicity studies and antigenic cartography to test hypotheses regarding the antigenic or neuroinvasive dominance of individual epitopes within the glycoprotein (i.e. exactly which part of the glycoprotein is responsible for the increased pathogenesis, cell susceptibility and host inflammatory responses and can we use this data to predict the pathogenic potential of emerging and novel lyssaviruses or act as a target for future anti-viral candidates ).

Objective 02 Construction and assessment of lentiviral pseudotype viruses for the study of lyssavirus glycoproteins. [36 months]

Objective: In collaboration with UCL, FLI and CDC, we will construct and assess lentiviral pseudotype viruses containing the glycoprotein of EBLV-1, EBLV-2 and the emerging bat lyssaviruses respectively. The pseudotype viruses will be used to determine the cross neutralisation of bat lyssaviruses.

Rabies is one of the most deadly infectious diseases known with a case-fatality rate approaching 100%. Transmission most commonly occurs following a bite from an infected dog and annually >55,000 people die from this disease, the majority being located in the developing world. Hence, the most cost effective approach to eliminating the global burden of canine-mediated human rabies is to control canine rabies. In any species where vaccination may be applied, the ability to detect virus neutralising antibodies is pivotal to the evaluation of vaccination status. The currently prescribed virus neutralisation tests are expensive, time consuming and involve manipulations with live rabies virus. As such they are not readily performed in resource limited settings. To overcome this, lentiviral pseudotype neutralisation assays have been applied as an alternative platform for post vaccinal assessment of domestic animals and serosurveillance in bat species. The assays use a relatively small amount of serum, do not require BSL3 facilities and can be tailored to a particular laboratory through flexibility in the reporter detection system. We have developed a panel of pseudotype viruses representing a wide range of the currently recognised lyssaviruses, providing a tool set for serological surveys. The pseudotype viruses have been shown to be stable for up to 7 days at room temperature. Several serosurveillance studies and an inter laboratory ring trial between rabies reference laboratories have shown the RABV pseudotype assay to be a reliable and reproducible alternative to live virus neutralisation assays. The small quantity of serum required to perform this test, compared to standard assays, renders it particularly suited to serological surveys in bats. Pseudotype neutralisation assays will have widespread applicability for a range of serological surveillance studies (including those planned for the newly isolated Bokeloh bat lyssaviruses in UK, France and Germany and IKOV in Tanzania), to identify reservoir hosts, determine lyssavirus prevalence or assess post vaccinal seroconversion rates during rabies control programmes.

Objective 03 Application of antigenic cartography to study the cross neutralisation and antigenic profiles of the recombinant lyssaviruses and pseudotype viruses. [36 months]

Objective: In collaboration with Cambridge University, FLI and CDC, we will study the antigenic profiles of the recombinant viruses constructed in Objective 01 and the pseudotype viruses constructed in Objective 02. A representative panel of pseudotype viruses will be selected and validated for use in future antigenic cartography studies of lyssaviruses, vaccine strains and therapeutic reagents under low containment.

Although we have well established molecular techniques for quantifying genetic differences between viruses, it is arguably the antigenic differences which are more important when predicting the likely success of pre- and post-exposure rabies treatments, or assessing the risk posed by emerging lyssaviruses; a host’s immune system will see the antigenic properties of a virus, and not the genetic sequence. Antigenic variation amongst lyssaviruses is, however, poorly understood. Previous attempts to quantify antigenic difference have been limited by the low resolution and qualitative nature of antigenic data (i.e. presence or absence). Antigenic cartography can be used to make quantitative pictures of binding assay data, known as antigenic maps. This project has extended our research activities to focus on resolving a larger number of viruses, representing all members of the lyssavirus genus. We therefore have a robust framework with which to further explore the relationship between genetic and antigenic variation among lyssaviruses, including the new and emerging lyssaviruses. Passive immune globulin use is a crucial component of rabies post exposure treatment, and we demonstrated that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses using antigenic cartography. Predicting antigenic distance in this way not only represents a fundamental breakthrough in the understanding of the relationship between genotype and phenotype, but will be a hugely practical tool for assessing the risk posed by emerging lyssaviruses.

Objective 04 Phylogenetic analysis of Ikoma Virus [36 months]

On May 11, 2009, an African civet (Civettictis civetta) displaying clinical signs consistent with rabies was


killed by rangers in Ikoma Ward within Serengeti National Park (SNP). The infected material was submitted to APHA for molecular analysis. Initially, the results of the phylogenetic analysis of partial nucleoprotein gene sequences from representative Lyssavirus species provided strong empirical evidence that the civet isolate represented a new lyssavirus species, designated Ikoma lyssavirus (IKOV). Using next generation sequencing (Roche 454 platform), we have successfully generated full genome sequence for IKOV and confirmed the inclusion of this virus as a novel and highly divergent lyssavirus species. The sequence diversity from RABV vaccine strains suggests that currently available vaccines may not protect an individual from infection with IKOV. Indeed, the occurrence of the IKOV case in a rabies free area of the SNP undergoing a sustained rabies vaccination control programme supports this belief. Cross protection and cross neutralisation studies are underway for this novel virus to determine the likely success of rabies vaccines should humans or pets be exposed to this virus. Serological surveillance in a range of species (including bats) in Tanzania, e.g. using an IKOV pseudotype virus (under construction), may confirm if the African civet is the reservoir host of this lyssavirus or simply an incidental host.

Project Report to Defra8. As a guide this report should be no longer than 20 sides of A4. This report is to provide Defra with details of

the outputs of the research project for internal purposes; to meet the terms of the contract; and to allow Defra to publish details of the outputs to meet Environmental Information Regulation or Freedom of Information obligations. This short report to Defra does not preclude contractors from also seeking to publish a full, formal scientific report/paper in an appropriate scientific or other journal/publication. Indeed, Defra actively encourages such publications as part of the contract terms. The report to Defra should include: the objectives as set out in the contract; the extent to which the objectives set out in the contract have been met; details of methods used and the results obtained, including statistical analysis (if appropriate); a discussion of the results and their reliability; the main implications of the findings; possible future work; and any action resulting from the research (e.g. IP, Knowledge Exchange).

All Figures are provided in the accompanying Appendix

Objective 01 Application of Reverse Genetics Technology to study the role of the EBLV proteins in viral pathogenicity, neuroinvasiveness and immune evasion. [35 months]

All milestones met

The UK is free from classical rabies but a closely related variant, European bat lyssavirus type 2 (EBLV-2), is endemic in a species of UK insectivorous bat and was responsible for the death of a bat conservationist in Scotland in 2002.

As the first step in a process aimed at identifying the viral genetic elements responsible for EBLV pathogenicity, two infectious recombinant viruses (vaccine strain SAD-B19 backbone containing the glycoproteins of EBLV-1 and -2) have been successfully recovered from cloned cDNA under Seedcorn project SC0163 and assessed. The SC0163 study involved the successful technology transfer from experts in the field of reverse genetics, Professors Bernhard Dietzschold and Dr Matthias Schnell.

A study in the laboratory of Professor Schnell identified viral genomic elements responsible for the differences in neuroinvasiveness between a silver haired bat virus strain and an attenuated vaccine strain. By creating infectious recombinant viruses they were able to study the genetic regions associated with the viruses’ ability to invade the CNS from a peripheral site. In addition to the targeted exchange of genes between neuroinvasive and non-neuroinvasive lyssaviruses, infectious rabies clones have been used as vectors to express foreign genes and in the development of novel rabies vaccines.

This project planned to build upon the skills acquired at Thomas Jefferson University and enable the research and technology to be further developed at the AHVLA. The study involved the construction of an infectious EBLV-1 within which the EBLV genes can be replaced by respective genes from other lyssaviruses. The construction of the EBLV-2 infectious clone backbone and helper plasmids was delivered under a Seedcorn grant (SC0242). The recombinant viruses were compared to their wild type strains in-vitro (tissue culture studies, growth characteristics, cell line susceptibility) and in vivo (mouse model) to facilitate a greater understanding of the role played by EBLV proteins in pathogenicity and neuroinvasiveness.


M01/01 Submit a report on the use of a SADB19 infectious clone to study EBLV glycoproteins (9 months)

Under the Seedcorn project SC0163, in collaboration with Professor Schnell, the G gene of each EBLV was cloned to replace the parenteral G gene of a previously characterised rabies infectious clone backbone (vaccine strain SAD B19). The recombinant (chimeric) viruses (cSN-EBLV-1G and cSN-EBLV-2G) were recovered from cloned cDNA by transfection of BHK derived cells (Figure 1) and the influence of EBLV glycoprotein in this non-neuroinvasive RABV backbone was assessed in vivo and in vitro.

The following extract is taken from the report prepared for the SC0163 infectious clone studies (fulfilling milestone M01-01).

After successful rescue of cSN-EBLV-1 and cSN-EBLV-2 viruses, titrations were performed with the wt-EBLV-1 and -2 and cSN using the TJU methodology resulting in ffu/ml and also the AHVLA method resulting in infectious virus particles/ml (see Table 1). The titration results show overall comparison between the methodologies, although the AHVLA results were up to 1 log higher than achieved using the TJU methodology. This is due to the AHVLA method recording individual cells as positive, whereas the TJU method only records a foci of infected cells. In order to have comparable results with the TJU published papers we used the ffu/ml results to perform the in vitro and in vivo experiments

. Table 1: Titration results for the viruses using TCID50 and ffu/ml protocols

In Vitro Growth Characteristics. The addition of 1 virus particle per cell (MOI 1) and taking time points up to 96 hours enabled us to study the basic growth kinetics of the viruses (MOI 1 single-step growth curve). The analysis of the time points by titration is shown in Figure 2a. Apart from the cSN early time points (0 and 6 hours, due to cell stripping) all titrations were successful. The growth kinetics of the cSN and wt-EBLVs varied considerably with cSN at times having a titre 100-fold higher. Within 6 hours all viruses (except wtEBLV-2) were detected in the supernatant. During the first 18 hours the cSN-1 recombinant titres were comparable to the cSN backbone virus, 400-fold higher than wtEBLV-1. The titre of all viruses (except wtEBLV-2) levels off after 48 hours. The endpoint titre of wtEBLV-1 and cSN-1 were very similar (within 3 fold), 100-fold lower than cSN.

The addition of 0.01 virus particles per cell allows the study of viral growth rate and spread from cell to cell (multi-step curve). The multi-step growth curve (Figure 2b) clearly demonstrated how the cSN-1 recombinant virus kinetics mimic the cSN backbone in the first 24 hours PI with the wtEBLV-1 growth delayed until 18 hours PI. By 72 hours the cSN-1 recombinant virus titre is equivalent to wtEBLV-1, 25-fold lower than cSN backbone. Both the single- and multi-step growth curves reveal that the early stage events of virus growth and spread do not involve the glycoprotein gene, and that the cSN backbone genes enable the virus to obtain higher titres than the wt-EBLVs during the early stages of growth. However, the glycoprotein is crucial in determining the end point titre. We hypothesise that the amount of glycoprotein generated in the wtEBLV-1 and cSN-1 is lower than in the cSN backbone limiting the final amount of virus released into the media.

In vivo MLD50 titrations were carried out with two routes of inoculation, intracranial (IC) and footpad (FP) for each virus. The IC MLD50 challenge results for all viruses showed a clear dose-dependent effect, with both a delay in time to mortality and also an increase in the number of survivors. This dose-dependent effect was only observed in the FP MLD50 results for the wt-EBLVs, but not for the recombinant viruses where only the undiluted and 1:10 dilution resulted in a low level mortality. We intended to use these MLD 50 results to determine appropriate virus concentrations for the Neuroinvasiveness experiment. However, the footpad MLD50 calculations for the recombinant viruses (cSN and cSN-1) could not be determined (even undiluted virus did not cause high mortality) therefore equivalent virus particles using the ffu/ml calculation was employed either by using the MLD50 dilution most closely related to the value, or by using the highest virus concentration available (undiluted).


Virus VLA log 10 INV log = virus/50ul

VLA virus/ml (x 20)

Ffu/ml TJU

RV9 4.8 63,095 1.2 x10 6 5.0 x 10 5 RV1787 3.9 7,943 1.6 x10 5 5.0 x 10 4 CSN - 1 5.5 316,227 6.2 x10 6 1.6 x10 6 CSN - 2 2.7 501 1.0 x10 4 6.1 x10 3 CSN 5.8 630,957 1.2 x10 7 6.6 x10 6

In vivo Neuroinvasiveness experiment; all viruses, when inoculated with 100ffu IC, resulted in 100% mortality, usually within 24 hours of the first mortality. The number of days post inoculation mortality was first observed, varied between the viruses in the order wtEBLV-1 (5 DPI)<cSN-1 (6 DPI)<cSN (7 DPI)<wtEBLV-2 (9 DPI). The difference between the wtEBLVs has been previously observed and is significant, the one day difference each between wtEBLV-1, cSN-1 and cSN is too short to draw any significant conclusions. The cSN backbone has previously been shown to be non-neuroinvasive (unable to cause mortality when inoculated in a peripheral site), therefore, the footpad neuroinvasiveness experiment was performed to determine if partial or complete reversion of this phenotype, by replacing the cSN glycoprotein with that of wtEBLV-1, was possible. 3x104 ffu (or highest possible titre) of each virus was inoculated in the footpad of each mouse and observed daily for clinical signs (Figures 3 and 4).

Similar to the IC results, the wt-EBLV-1 infected mice were the first to show clinical signs (6 DPI) with an overall survivorship value of 60% which was the lowest survivorship value we obtained for the virus. In contrast, only one mouse died from the cSN challenge (confirmed by FAT and RT-PCR as positive) on day 10 PI resulting in a 90% survivorship. Interestingly, cSN-1 also had the first mortality on day 10 PI, finishing with a survivorship rate of 60% identical to the wtEBLV-1, indicating that the wtEBLV-1 glycoprotein is the major determinant for neuroinvasiveness.

Histopathology (Figure 5); Analysis of these samples was performed the results are summarised in Table 2. Histopathology of brains from both intracranial and footpad inoculated mice showed inflammatory changes induced by the viruses in order of severity wtEBLV-1<wtEBLV-2<cSN-1<cSN, with cSN inducing severe inflammatory changes. Interestingly only IC animals had hippocampal necrosis. In contrast to the inflammatory changes, for both sites of inoculation, the number of immuno-labelled neurons in the brain (detection of virus) was wtEBLV-1>wtEBLV-2>cSN-1³cSN. Animals showing more severe inflammatory changes in the brains (cSN and cSN-1 inoculated) seemed to display fewer immunolabelled neurons. This increased inflammatory response may be partially responsible for limiting the spread of the virus, inducing destruction of infected neurons, or be just the consequence of neuronal destruction by the virus, triggering local inflammation as the cellular debris are cleared by local microglial cells undergoing activation and inducing chemotactic factors.

Table 2 Brief summary of histopathological and immunohistochemical observations. Histology (severity of histopathological changes): +/- minimal, + mild, ++ moderate, +++ severe. Antigen (number of immunolabelled cells): +/- minimal, + few, ++ multiple , +++ abundant/widespread

In the spinal cords of the IC inoculated mice, there were no histopathological changes observed for all viruses although virus spread from the brain into the spinal cord was observed for all viruses examined (wtEBLV-1, -2 and cSN-1). The severity of the pathological changes observed in the spinal cord following FP inoculation of the different viral strains seems to be more prominent with cSN and cSN-1, with prominent


Inoculation Route Tissue Feature RV9

WtEBLV-1 cSN1 cSN RV1737WtEBLV-2

Intracranial

Encephalon

Histology(lesions/inflammation)

+/- ++ ++ +

Antigen (IHC) ++/+++ to +++ + to ++ ++ ++ to +++

Spinal CordHistology - - - -

Antigen + +/++ + +/++

Footpad

Encephalon Histology + ++ to +++

++ to +++ ++

Antigen +/++ to +++ +/- +/- + to +/++

Spinal CordHistology + to ++ +++ +++ ++ to +++

Antigen + to ++ +/- - +

glial nodule formation. wtEBLV-1 and wtEBLV-2 elicit a milder and diffuse microglial activation, slightly more marked in wtEBLV-2 with evident lymphocyte infiltration. In general terms, despite the substitution of the glycoprotein gene of cSN by wtEBLV1, the histopathological changes and viral antigen distribution, as shown by immunohistochemistry, are closer and very similar to the original cSN virus than to the wtEBLV-1strains from which the glycoprotein gene was obtained.

All clinical scores correlated with FAT/RT-PCR results with the exception of one mouse in cSN FP group which had hind leg paralysis and was killed 19DPI yet was subsequently shown to be FAT and RT-PCR negative. All but 5 FAT and RT-PCR results correlated (were all FAT negative, PCR positive - 1 x RV9 IC and 4 x cSN-1 FP).

Objective 1 Conclusions

Factors affecting the replication of EBLVs are poorly understood. Isolation of EBLVs has demonstrated that they are generally unable to replicate efficiently in cell culture compared to RABV strains. Whilst RABV isolates generally replicate to titre exceeding 105 EBLVs generally reach much lower titres (<104.5) in comparative cell culture systems. To test mechanisms that may restrict infection and replication within cell culture we set about using reverse genetics to assess the role of the viral glycoprotein in cell entry and replication in vitro and in vivo to determine whether swapping glycoproteins from EBLVs into a RABV system would reduce the titre of the recombinant viruses. The generation of recombinant RABVs containing heterologous G proteins has shown that RABV can tolerate interspecies G substitution. The SN full-length RABV clone was manipulated such that G was replaced with that from either EBLV-1 (SN-1) or EBLV-2 (SN-2). These recombinant RABVs were able to grow in both in vitro and in vivo models, confirming their ability to replicate successfully and spread between cells. Alongside this we assessed whether swapping the G protein between RABVs and EBLVs in the recombinant RABV backbone affected antigenicity. Antigenic cartography was used to measure the antigenic effect of replacing G in SN-1 and SN-2 by placing them on an antigenic map alongside other RABV, EBLV-1 and EBLV-2 viruses. SN-1 and SN-2 were antigenically indistinguishable from their wild-type counterparts (EBLV-1 and EBLV-2, respectively), and distinct from SN (RABV), confirming that G is the dominant antigenic component of lyssaviruses, as determined by neutralizing antibodies. These analyses also demonstrated the potential to use reverse genetics and antigenic cartography to test hypotheses regarding the antigenic dominance of individual epitopes within the G.

Replacing the entire G ORF (as opposed to just the ectodomain) has enabled investigation of the function of the EBLV G in an RABV backbone. Amino acid divergence between G may suggest that proteins from each species have evolved to interact optimally, therefore with only 70.3 % (CVS : EBLV-1) and 74.3 % (CVS : EBLV-2) amino acid identity, cross-species protein interactions could be affected. Indeed, the in vitro results indicate that SN-1 and SN-2 decrease in fitness compared with the homologous SN virus in both single- and multi-step time-course experiments. The reduction in virus titre was evident from not only the peak titre reached by each virus, but also the titre at each time point. However, it is important to analyse these data alongside the growth kinetics for each of the respective EBLVs. Both SN-1 and SN-2 improved their viral growth curve kinetics in comparison to the EBLVs, indicating that the EBLV G is able to interact with heterologous RABV M protein during encapsidation and budding. Moreover, this suggests that the EBLV G is not the dominant protein influencing virus titre in EBLVs, as the recombinant viruses grow to equivalent (SN-1), or higher (SN-2) titres than wild type EBLV-1 and EBLV-2, respectively. This observation would indicate that the low titres observed for EBLVs are not a result of G-dependent processes such as receptor binding or entry but of G-independent steps of virus replication.

The importance of G for neuroinvasion was investigated by replacing the G gene of SN, with a G gene from two neuroinvasive lyssaviruses and inoculating both via ic and peripheral (fp) routes. The pathogenic ability of the recombinant and backbone viruses in relation to the wild-type viruses was confirmed by ic inoculation. However, to study the effects of G via a route mimicking natural exposure, a peripheral challenge was also undertaken. The SN, SN-1 and SN-2 Kaplan–Meier survival curves show that there is a threshold, under which all animals survive a peripheral challenge, and over which one or two animals may succumb, rather than a dose-dependent response. Because SN is an attenuated, cell-adapted virus, inoculation of higher doses of SN, SN-1 and SN-2 was possible. For SN, a high dose (higher than achieved for any wild-type EBLV) was required to observe any clinical signs, in one (20 %) of the animals. In contrast, the EBLVs are not cell-culture-adapted and are pathogenic at lower doses. The EBLV titration resulted in animals succumbing to as little as 10 ffu (2 log10 lower than the rescued viruses), a result that may reflect the selective pressure on wild-type viruses to minimize replication to evade the immune system while increasing pathogenicity and neuroinvasiveness. By swapping G between laboratory adapted and wild-type lyssavirus isolates, we have addressed the significance of G in neuroinvasiveness and pathogenicity. All viruses were compared at 103 ffu. in a peripheral in vivo challenge. At this dose, there was no increased neuroinvasiveness observed in SN-1 and SN-2 over SN. Only the EBLVs resulted in clinical disease significant in comparison to the recombinant viruses (P=0.01 EBLV-2 and P=0.04 EBLV-1). The SN-1


peripheral challenge study at 104 ffu resulted in 73 % survival, which is not significantly different from EBLV-1 (P=0.18). In contrast, the difference observed between the SN and SN-1 Kaplan–Meier plots is significant (at the 90 % CI level; P=0.06), indicating the G substitution has an effect on the neuroinvasive properties of the SN virus. Regrettably, SN-2 could not be compared at this high titre. The involvement of other viral proteins remains unclear, but M has previously been shown to support budding and therefore play a role in virus cell-to-cell spread. Thus it appears more likely that a combination of differences across the viral genome, rather than a single gene, contribute to the attenuated phenotype of the SAD-B19 strain.

Histopathological analysis was undertaken on the clinical animals from both the ic- and fp-inoculated groups. In the ic-inoculated mice, all viruses spread from the brain into the spinal cord and also to the neuronal bodies of the sensory neurons (DRG) in the peripheral nervous system with equal ability. For the fp-inoculated mice, SN and SN-1 resulted in more severe inflammatory changes in the brain and appeared to display fewer immunolabelled neurons than EBLV-1. This increased inflammatory response may be partially responsible for limiting the spread of the virus, or just the consequence of neuronal destruction. The higher survival rate of the mice inoculated peripherally with the recombinant viruses over the wildtype viruses supports this observation, suggesting that there is an inverse correlation between the neuroinvasiveness of the virus and the inflammation observed. For both routes, despite the G present, the histopathological changes and viral antigen distribution observed for SN-1 and SN-2 were more representative of those seen with the SN virus than the EBLVs. This indicates that the host response is more heavily influenced by other viral proteins important in the development of the histological lesions observed. The manipulation of a RABV backbone virus (SN) to rescue recombinant viruses with complete G from two different lyssavirus species has demonstrated successfully that G is the dominant target for neutralizing antibodies. Furthermore, we have shown that interspecies G substitution reduces the virus titre in comparison with SN. Future work investigating the cytoplasmic domain of G may further define restrictions to interactions between G and M and/or RNP. Our observation that SN-2 end titre increased significantly (P<0.001) in comparison to EBLV-2 indicates that G-independent factors are responsible for the low titre levels observed for EBLV-2. The presence of G from a neuroinvasive virus in a non-neuroinvasive backbone appears to help the virus reach the CNS in the case of SN-1, albeit only at a high virus titre.

Future work may involve the constructs being manipulated further to identify specific regions of G that are responsible for the neutralizing antibodies and cell entry.

M01/02 Construct EBLV-1 infectious backbone and helper plasmids (20 months)

Under ROAME project SE0420, we obtained complete genome sequence data for a German EBLV-1 isolate. This data facilitated the construction of an EBLV-1 backbone within which the EBLV genes can be substituted with the respective genes from other lyssaviruses including the non-neuroinvasive SADB19.

In house construction of the backbone can be time consuming and lengthy and involves multiple PCR and ligation stages followed by genome sequence confirmation. The backbone was instead sourced externally via customised nucleic acid synthesis.

Prior to synthesis of the backbone a restriction mapping exercise was performed to enable design of a full length non-infectious clone. To this end all available full length lyssavirus genomes were screened for restriction endonucleases (REs) not present within them. This highlighted a number of RE sites that could be carefully positioned within the genome construct to enable swapping of genes between isolates and ultimately, genotypes. Furthermore, the backbone vector used for expression of the full length genome was screened for the same reason. The full list of REs that were not present in any of the genomes and vector backbone is shown below:

AscI, AsiSI, FseI, PmeI, SbfI, SgfI, SgrAI, Sse232I

From this list, only 5 restriction enzymes were of use for cloning. Previous experience with manipulating full length genomes was used to identify the optimal enzymes. However, the insertion of 6 restriction sites were required to enable manipulation of all 5 lyssavirus open reading frames (ORFs) and so another enzyme was chosen based on its cut site within other lyssavirus genotypes. The requirements for this project and further downstream projects were that swapping of the matrix protein (M), the glycoprotein (G) and M and G in tandem was possible. For this reason PacI was also selected for inclusion in the new full length clone as although this enzyme cuts genotype 3 lyssavirus isolates (Mokola) it cuts at position 4587 (in EU293118) so as long as it wasn’t positioned as an enzyme used to manipulate M and G then its inclusion would not be problematic.

Furthermore, to allow swapping both M and G individually and in tandem, restriction sites that cut leaving non compatible overhangs were applied to the untranslated regions flanking each of these ORFs. The final


plan regarding choice and positioning of REs within the EBLV clone are detailed in Figure 6. Restriction sites were positioned to try and avoid disruption of highly conserved sequences present at gene junctions. However, the requirements of sequence at each of the gene junctions is largely unknown and so disruption of potentially important sites cannot be predicted. Only successful virus rescue and growth kinetics compared to the wild type virus will indicate if the sites have not disrupted viral transcription and/or replication. The T7 promoter/ T7 termination sequences and ribozyme sequences required for expression and rescue of virus from the full length clone were used as present in the rabies SAD B19 developed through SCO163. Full length sequence including restriction sites and promoter sequences were sent to the US based company DNA20 for synthesis commercially.

Unfortunately, progress was significantly hampered by delays in the commercial generation of the full length EBLV clones. Furthermore, when finally generated, toxicity of the plasmids to the E. coli cell line used for amplification has made generation of DNA from which to perform rescue very difficult. The commercial manufacturer recommended different strains of E coli for DNA preparation but stated that they had strong concerns regarding maintenance of the plasmids in bacteria as deletion mutants were readily generated during the synthesis project - an aspect that delayed the supply of the clones. We investigated several different bacterial strains to assess growth of these plasmids. Both the EBLV-1 and -2 full length plasmids required growth at room temperature to minimise deletions within the sequence. Following optimisation we were able to generate DNA in Stbl3 cells (a bacterial cell line often utilised where issues regarding mutation of input DNA are encountered) as well as in XL-1 Blue super competent cells. However, for both plasmids the growth requires room temperature amplification over a period of 5 days with low yields. Potential deletion mutants remained problematic.

Initial attempts with the heterologous CSN helper plasmids failed to rescue either EBLV-1 or -2 and so homologous EBLV-1 and -2 helper plasmids were obtained from FLI Germany to try and assess the new full length DNAs. We demonstrated successfully that the EBLV-1 helper plasmids are functional and were able to ‘rescue’ CSN virus.

The positive rescue of the CSN backbone with the EBLV-1 helper plasmids supplied by our collaborator gave us some hope for the rescue of EBLV-2. However, despite numerous attempts we were unable to rescue the synthesised EBLV-2. Furthermore, one of our collaborators has constructed and attempted to rescue an EBLV-2 virus but has also been unsuccessful. Currently, we do not have EBLV-2 specific helper plasmids that are functional and it appears that, despite earlier beliefs to the contrary, neither the EBLV-1 nor the CSN helper plasmids are able to rescue our EBLV-2 clone. This milestone had previously been extended by 6 months (CCF01). A considerable effort and expense had been invested in the design and synthesis of the EBLV recombinants and we did not wish to completely abandon this work at this late stage. However, there was little confidence in the success of this venture within the time frame of the project and as a further two milestones were dependent upon the rescue of the recombinants (M03/03 and M01/04), we proposed (CCF02) to amend milestone M01/03 and remove the milestones M03/03 and M01/04. In an attempt to understand why we were experiencing difficulties we hoped to sequence relevant regions of the backbone constructs (if possible using 454 sequencing). Hence we proposed, that milestone M01/03 was altered to the following: M01/03 – Sequence the EBLV-1 and EBLV-2 backbones due 28-02-12 –met

Following the initial problems encountered with both the growth of the full length genome plasmids in different bacterial stocks and the inability to rescue live virus from either clone (EBLV-1 or EBLV-2), we acquired full genome sequence data of the small scale plasmid preparations that we had generated using the 454. Whilst the EBLV-2 clone appeared to be the correct sequence, a short deletion was identified within the vector backbone sequence of the EBLV-1 clone. Unfortunately, the effect of this deletion on the ability to rescue the clone remains unknown.

02 Construction and assessment of lentiviral pseudotype viruses for the study of lyssavirus glycoproteins. [36 months]All milestones met.Neutralising immune responses to RABV are directed against the viral Glycoprotein (G). Current vaccines are based on this knowledge and comprise inactivated virus presenting G on the surface which stimulates an immune reaction. A more extensive monitoring of humoral responses to RABV vaccines would allow for a more specific and proportionate response in endemic countries. However, given that that majority of these countries have resource limited laboratories and that the routine method to test for RABV Virus neutralising antibodies (VNAs) involves BSL3 facilities, it is not practical to carry out thorough and sustained surveillance. Retroviral pseudotypes are virus particles with a packaged reporter gene, retroviral Gag and Pol core proteins and an outer envelope derived from another viral source (Figure 7). These viral pseudotypes are a very powerful tool for molecular biologists as typified by the large number of examples in the literature of retroviral pseudotypes being used in research for gene therapy, cell entry and receptor


identification and virus neutralisation. Our collaborators at University College London (UCL) have previously shown that pseudotyped retroviral particles expressing the envelope proteins of SARS CoV and H5N1 influenza can be used to develop assays that are sensitive and specific for the detection of viral neutralising antibodies (VNAs) to these pathogens. This high level of efficacy coupled with the removal of a need for high containment facilities, and the small volume of sera needed for tests, make pseudotypes very attractive for serological surveillance.

Lyssavirus Pseudotypes

We have proof of concept regarding the development of a RABV pseudotype neutralisation assay (published in Journal of General Virology in 2008 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2886951/). The CVS-11 pseudotypes can be employed for the detection of RABV VNAs. The pseudotype assay will aid three aspects of lyssavirus serological surveillance. Firstly, the assay can be used safely, without BSL3 facilities, in studies to accrue the prevalence of natural lyssavirus infections in domestic and wild species. Secondly, the assay can be used locally where large scale field trials of RABV vaccines are being tested in dogs. Thirdly, the prevalence of lyssavirus in European bat populations can be determined using pseudotypes of different European Bat Lyssavirus (EBLV) genotypes. Luciferase reporters required expensive light-emitting reagents and GFP requires fluorescent microscopy. Therefore, in order to render the pseudotype assay easy and inexpensive to apply in resource poor countries, we constructed pseudotypes with a β-galactosidase (β-gal) reporter gene. β-gal staining with X-gal reagent can be simply measured in an ELISA plate reader or even by eye (Figure 8). Such an assay will be practical in countries lacking BSL3 high containment facilities or specialized equipment. The use of lyssavirus pseudotypes would have significant benefits for micro-serological analysis of bats.

Virus neutralization assays such as the FAVN, RFFIT or modified derivatives require at least 40μl of sera, which is usually the total volume of sera sampled from small species such as insectivorous bats. Hence, there is a generally insufficient serum for confirmatory or repeat testing. Subsequently it is difficult to share sera for quality assurance or reference purposes. The pseudotype neutralisation assay (PNA), utilises relatively lower volumes of sera (~10μl), allowing for multiple testing. Studies to determine the prevalence of emerging lyssaviruses in bat populations can thus be enhanced using pseudotypes of different virus species.In collaboration with UCL, we constructed and assessed lentiviral pseudotype viruses containing the glycoprotein of RABV, EBLV-1, EBLV-2, DUVV, LBV, MOKV and the Eurasian lyssaviruses ( fulfilling the milestones M02-01 and M02-02). The glycoproteins of the emerging lyssaviruses (Irkut virus, Khujand, Aravan and West Caucasian Bat Virus) were synthesised from published Genbank sequences by a commercial company. Preliminary assessments (Figures 9 & 10) were carried out using the pseudotypes including verifying the antigenic properties of the fully synthesised Eurasian lyssavirus pseudotypes in modified FAVN assays (using sera raised in rabbits against the Eurasian viruses at CDC Atlanta). The LBV, MOKV and WCBV PNAs were then employed in a study of African fruit bat (Eidolon helvum) sera in Ghana. Of the bat sera screened 56% neutralised ptLBV, 27% neutralised ptMOKV and 1% neutralised ptWCBV, supporting the hypothesis that LBV is endemic in Ghanaian E. helvum. Serum VNAb titres were determined using the PNAs and modified FAVN assays (for LBV & MOKV) and a high correlation (r), sensitivity (Sn) and specificity (Sp) was observed between the two approaches. The study was published in 2010 (http://www.sciencedirect.com/science/article/pii/S0042682210006021#)

RABV Pseudotype

Additional validation data has been obtained for the RABV pseudotype (CVS-11) including achieving excellent correlation between the pseudotype assay and the Fluorescent Antibody Virus Neutralisation (FAVN) assay (Figure 11) using archived PETs sera (post vaccination pet dog and cat sera). The RABV pseudotype assay has also been applied to a sero-surveillance study of vaccinated dogs in Tanzania. We analysed 304 serum samples from Tanzanian dogs for the detection of rabies antibodies in a pseudotype assay using the CVS-11 PNA. Compared with the FAVN assay, a specificity of 100% and sensitivity of 94.4% with a strong correlation of antibody titres (r = 0.915) were observed with the pseudotype assay (Figure 12). Importantly, the CVS-11 pseudotypes were highly stable during freeze–thaw cycles and storage at room temperature (Figure 13). These results suggest the proposed pseudotype assay is a suitable option for undertaking lyssavirus serosurveillance in areas most affected by these infections and were published in Vaccine in 2009 (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789314/).

CVS-11 Pseudotype Neutralisation Assay (PNA) Ring Trial


http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2789314/

http://www.sciencedirect.com/science/article/pii/S0042682210006021#


The sero-surveillance studies provided confidence in the ability of the RABV PT to assess post vaccinal VNAs. The observed sensitivity of the PNA when testing the Tanzanian dog sera was 94% compared to the FAVN but this allowed for 100% specificity. Discordant PNA titres were observed for a small number of the Tanzanian dog sera with VNAs around the FAVN cut-off of 0.5IU/ml. To assess intra- and inter- laboratory reproducibility of the CVS-11 PNA, we have undertaken a ring trial of the CVS-11 PNA. This study will enable us to assess our ability to transfer the CVS-11 PNA to other rabies laboratories, equally equipped and experienced in performing rabies virus neutralisation assays. We invited six reference laboratories to participate but unfortunately, difficulties with legal or Health & Safety requirements (MTA or GM) prevented three labs from receiving the ptCVS-11 (Table 3).

Ref Lab Country Admin Requirements

Results Provided

AHVLA UK Cleared YesAMMS China Not Cleared NoANSES France Cleared YesCDC US Cleared YesCFIA Canada Not Cleared NoFLI Germany Not Cleared NoKSU US Cleared YesOVI South Africa Cleared Yes

Table 3. Laboratories invited to participate in the CVS-11 Pseudotype Ring Trial

A panel of 6 dog sera were tested by FAVN in triplicate at AHVLA (Table 4). The sera were blinded and issued to each participating lab with a stock of ptCVS-11, a protocol and batch sheets. Test and control sera were titrated in duplicate in doubling dilutions in a 96 well microtitre plate before a constant dose of ptCVS-11 virus was added. The plate was then incubated at 37°C for 60 minutes. This allowed the antibodies in the sera to bind to their target antigen (the rabies envelope G-protein) and therefore inhibit infection. After incubation of the serum/pseudotype mix, a suspension of BHK cells was added and incubated at 37°C for a further 48 hours.

Table 4 Replicate FAVN titres for the blinded panel of sera (samples 1-6)

The CVS-11 pseudotype chosen for the trial carries the reporter gene LacZ, therefore any non-neutralised pseudotype virus is indicated by a blue-stained nucleus within the cell (Figure 14). The titre of the test serum sample is calculated based on the highest dilution where 100% neutralisation is observed and the number of neutralised wells at and beyond that dilution (Spearman Karber Method as for FAVN)

The panel included three borderline sera, two high positive sera and a negative serum (naïve dog). The results are blinded and shown for the five participating laboratories (Table 5). The titres obtained using the CVS-11 PNA indicated inter-laboratory reproducibility. All laboratories could discriminate between their negative and positive controls. Overall, the concordance observed was good. Discordant results (highlighted) were obtained by laboratory D, particularly for samples S2 and S3 (perhaps the samples were accidently switched?). Laboratory A generally observed higher titres for the samples tested but did discriminate between the negative dog sera (Sample 3) and the other sera. For Labs A-D, the borderline sera yielded anomalies in the PNA, but such variability was equally observed in the FAVN titres. Four of the five labs correctly identified the negative dog sera (sample 3) and also discriminated the high and borderline sera for all samples.

FAVN Run A1 A2 B1 B2 C1 C2 D1 D2 E1+ve Control 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50-ve Control 0.05 0.05 0.09 0.09 0.13 0.09 0.13 0.25 0.25S1 borderline 4.00 4.00 0.50 0.35 0.50 0.50 0.25 2.00 0.50


FAVN Run 1 2 3 Mean Titre CommentSample 1 1.14 0.87 0.50 0.84 BorderlineSample 2 276.96 210.40 276.96 254.77 High positiveSample 3 0.03 0.04 0.05 0.04 Dog negativeSample 4 159.90 276.90 92.31 176.37 High positiveSample 5 0.66 0.38 0.66 0.57 BorderlineSample 6 0.22 0.38 0.29 0.30 Borderline

S2 high +ve 1448.15 724.00 90.51 128.00 362.04 181.02 0.25 0.50 362.04S3 Dog -ve 0.05 0.05 0.09 0.06 0.13 0.09 11.31 1.00 0.25S4 high +ve 1448.15 724.00 128.00 90.51 362.04 256.00 64.00 256.00 362.00S5 borderline 2.83 2.83 1.00 1.00 0.71 0.35 0.25 2.83 0.50S6 borderline 0.71 0.50 0.35 0.35 0.50 0.71 1.00 2.83 0.50Virus Titration 512 724 256 128 16383 2048 22 8 128

Table 5 CVS-11 PNA titres obtained by the five participating labs (A-E). All but lab E performed the assay in duplicate.

For laboratories A-D, intra-laboratory comparisons could be made. Whilst there were some discordant results obtained by Laboratory D, intra-laboratory consistency was observed between replicate tests by most laboratories. Expected variations were observed for the borderline sera (as seen by FAVN).

The ptCVS-11 virus titres varied between laboratories. Laboratories A and C had particularly high virus titres, which may account for the slightly higher PNA titres obtained. Laboratory D had very low virus titres, which may have resulted in the anomalous results observed between runs and compared to other laboratories.

EBLV-1 and EBLV-2 Pseudotype Neutralisation Assay

Active surveillance for EBLV-1 and -2 (European bat lyssaviruses Type 1 and 2) and Bokeloh bat lyssavirus (BBLV) is performed in a number of countries across Europe using modified variations of the FAVN (mFAVN). However, there is no harmonised approach to testing. The use of different viruses, the absence of reference bat serum (unfeasible) and differences in result interpretation (cut-off values) make comparisons between national surveillance programmes problematic. In addition, the small volumes of sera collected during live capture and release programmes usually limits testing to one lyssavirus species. The ability of the pseudotype assay to employ relatively smaller volumes of serum compared to mFAVN, combined with the potential to multiplex the assays to detect antibodies against a number of lyssavirus species using different reporter detection systems, supports their use in bat serological surveys.

We have successfully constructed EBLV-1 and -2 pseudotypes with three different reporter genes (Renilla, Luciferase and LacZ). The firefly luciferase and renilla luciferase based pseudotypes have also been multiplexed to allow for simultaneous detection of either EBLV-1 or EBLV-2 antibodies in the same test serum. A strong correlation was also observed between standard and multiplexed EBLV- and –2 pseudotypes. The ptEBLV-1 (RV9) utilises the renilla luciferase reporter whilst the ptEBLV-2 (RV1787) uses the firefly luciferase reporter. The sera (n=21) used for validation covered a range of titres from dogs and rabbits, previously immunised with commercial RABV vaccines or EBLV-1 and -2 inactivated viruses respectively (Figure 15).

The recombinants were successfully transferred to AHVLA and concordant titres were observed in the PNA luciferase assays between the two laboratories (UCL and AHVLA). Prior to circulation for a wider ring trial we established standard operating procedures and a panel of anti-EBLV sera (polyclonal rabbit serum in lieu of reference bat sera). To allow for a widely accepted and cheap platform, we selected the LacZ reporter system for the EBLV-1 and -2 pseudotypes. We made several attempts to generate large batches of LacZ pseudotype viruses to reduce variability when distributing the viruses to participating laboratories. However, technical issues with rescuing sufficient volumes of the recombinant pseudotypes continued to disrupt the validation of the EBLV-1 and -2 pseudotype assays. An external expert and collaborator (Dr Ed Wright) visited our laboratory but failed to identify any possible causes for the low viral titre. Staff performed a number of experiments e.g. comparing reagent batches, suppliers and technical variables. There has been some recent success at rescuing higher titre EBLV pseudotypes. Once those pseudotype batches have been validated they will be distributed to collaborators in European ref labs. A detailed report on the CVS-11 and EBLV pseudotypes (ring trial data, reproducibility, sensitivity and specificity) has been prepared and distributed to the participants and the EC reference Lab for Rabies Serology (fulfilling milestone M02/04). The interim results were presented at SGM 2013 in the form of a poster and an oral presentation will be delivered at the CRL Serology Meeting in Nancy, October 2013.

Objective 2 ConclusionIn collaboration with colleagues at UCL and several rabies reference laboratories, we have constructed and


employed a number of lyssavirus pseudotype viruses which have proven potential in serosurveillance studies and for the assessment of humoral responses following vaccination. The ptCVS-11 pseudotype neutralisation assay demonstrates great promise as an alternative tool for detecting rabies specific virus neutralising antibodies in resource limited countries and facilities. In several studies employing post vaccinal sera (human, cat or dog) we have demonstrated excellent sensitivity and specificity compared to the OIE and WHO prescribed FAVN. Future studies will involve the further validation of EBLV and BBLV PNAs using field bat sera and a wider inter-laboratory ring trial.

03 Application of antigenic cartography to study the cross neutralisation and antigenic profiles of the recombinant lyssaviruses and pseudotype viruses. [36 months]All milestones met

A panel of pseudotype viruses have been generated and selected to represent the optimum pan lyssavirus coverage on the antigenic map. The titres of polyclonal sera raised in rabbits against different lyssaviruses determined by modified Fluorescent Antibody Virus Neutralisation (mFAVN) correlated well with those determined by the pseudotype (PT) assays for most lyssaviruses. The exceptions were WCBV and LBV, with only low numbers of detectable titres. The results will be reviewed to determine if additional pseudotypes should be synthesized to expand the coverage for e.g. WCBV. However, the development of a pseudotype for the newly identified highly divergent Ikoma virus (see M04/02 below) may enhance the coverage for WCBV.

There is sufficient variation within the genus to cause variable vaccine efficacy. Studies of genetic variation however, lack biological significance and studies of antigenic variation have low resolution. We addressed these issues by analysing serum virus neutralization data with a computational technique, antigenic cartography, to produce an antigenic map for a global panel of 25 lyssaviruses. Antigenic distances were compared with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from glycoprotein amino acid sequence, important differences between genetic and antigenic relationships were shown. These included antigenic differences between vaccine strains and wild-type rabies viruses.

Passive immune globulin use is a crucial component of rabies post exposure prophylaxis, and we demonstrated that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses using antigenic cartography. To fulfil milestone M03-01, the antigenic cartography data was published (http://jvi.asm.org/content/84/22/11841.long) . Rather than submit two antigenic cartography papers (principle and application), on ROAMES SE0423 and SE0424, it was decided to combine the data and submit to a higher impact journal. The paper outlines the principle, validation and applicability of antigenic cartography to rabies control and post exposure prophylaxis:

Further validation of the antigenic cartography system was undertaken, including a comparison of data generated when sera is raised to the lyssaviruses in different host species (rabbit, mice, human; Figure 16) and a comparison of the relationship between antigenic and genetic profiles (Figure 17). Comparison of multiple species using antigenic cartography showed antigenic distances measured using rabbit sera, corresponding to those determined by mouse sera, informing extrapolation of animal model data. Passive immunisation against rabies remains a critical part of post exposure prophylaxis. Antigenic distances between viruses based on neutralizing titres are directly relevant as the in-vivo potency of immune globulins correlates with their in-vitro neutralization titres. One of the strengths of antigenic cartography is that the difference in titres of each serum against a range of viruses are used to create target distances, and hence the positions of sera on the map are also determined by those titres. Therefore, if the titre of a serum is known against selected viruses, antigenic maps can be used to predict titres against all the other viruses on the map, to an average accuracy of approximately a two-fold dilution (without actually testing against those viruses). We have shown that HRIG neutralization titres correspond to the distances on antigenic maps and are therefore predictable.

We have shown that fluorescent antibody virus neutralisation (FAVN) test data can reliably quantify antigenic differences among lyssaviruses when combined with antigenic cartography [Horton et al 2010] and that despite obvious biological differences between live-virus and pseudotype viruses, the PNA data correlate well with FAVN test data for the laboratory adapted strain CVS-11 (Wright et al 2008). However, in order to rely on PNA data for quantifying antigenic differences among divergent viruses, it is essential to first show that antigenic differences measured using the two techniques are equivalent. Hence, we compared PNA data with FAVN test data using a panel of rabbit sera, to validate the use of pseudotypes for measuring antigenic differences.

Sera were raised in rabbits using beta-propiolactone inactivated tissue culture supernatant. The ability of


http://jvi.asm.org/content/84/22/11841.long

selected rabbit sera to neutralise a panel of divergent lyssaviruses was assessed using a modified fluorescent antibody virus neutralisation test (mFAVN) with a fixed dose of each virus (100 TCID50). 50% Endpoint dilutions were calculated using the Spearman-Karber method. The same sera were then also tested for their ability to neutralise a fixed dose of the equivalent viral pseudotypes, using a 100% endpoint.

Paired serum titres were compared directly (mFAVN test titre against equivalent PNA titre), and with antigenic cartography. Antigenic maps were made using first the mFAVN test data (Figure 18), and then the PNA data and quantitatively compared. Maps were overlain, and the difference in position of the viruses measured. Also, the distance from each virus to every other virus was measured for both maps, and compared.

The antigenic map in Figure 19 shows the relationships among the lyssaviruses measured using PT data compared with their relationships determined by FAVN test data. The average difference in position (i.e on average, the distance the viruses have ‘moved’ between the two maps is 2.7 (sd 1.3) antigenic units, AU equivalent to three two-fold dilutions in titre). This is high relative to the resolution of the maps (which is approximately one antigenic unit). Distances from each virus on the map to every other virus were also measured on both maps, and plotted against each other (Figure 20). The correlation is low (r=0.55), confirming the difference in relationships on the two maps. However, equivalent analyses for a map made using only CVS, EBLV1 and EBLV2 shows improved correlation between PNA and FAVN test maps (Figure 21). The average difference in virus position between maps is only 1.1 AU (sd 0.3). These analyses demonstrate that antigenic relationships among this panel of lyssaviruses are different using FAVN test and PNA data. The likely reasons for this are two fold. Direct comparison of data for each serum (Figure 22) demonstrates that titres for RV634 and to a lesser extent RV131 do not correlate well between FAVN tests and PT. Also, there are very few detectable titres against the most divergent lyssaviruses (LBV and WCBV) on both assays, making meaningful comparison between PT and FAVN test data difficult, and making their positions on the antigenic map less robust. This last problem is not unique to these analyses- as serological relatedness decreases, accurate assessment of the antigenic differences also decreases and we are therefore restricted to measuring distances at a lower resolution (Horton et al., 2010).

When maps were made using only the viruses with good correlation between tests- CVS, EBLV-1 and EBLV-2, the relationships between the viruses are very similar (Figure 21). This gives confidence to interpreting antigenic distances among phylogroup I viruses such as vaccines strains, the EBLVs, and the putative novel lyssavirus from Germany, Bokeloh bat lyssavirus (BBLV). In addition, in contrast to the rabbit sera data above, good correlation (0.83) was achieved for the LBV PNA (compared to an LBV mFAVN) using 184 field bat sera (Wright et al., 2010) when a relatively high cut-off was employed (<1:40 VNA).

Use of Antigenic Cartography for studying lyssavirus infectious recombinants

The recombinant viruses rescued under SC0163 were applied to the lyssavirus map. The clones cSN-EBLV-1G & cSN–EBLV-2G co-located with respective EBLV wildtype viruses and not with their backbone SADB19 virus (Figure 23). In addition mouse antisera obtained from in-vivo studies (SC0163) was assessed using antigenic cartography against a street RABV (RV61). Similar patterns were observed for the recombinants to those generated using rabbit sera raised against wildtype EBLV. This confirms the importance of G protein in the neutralisation of lyssaviruses. Table 6 is a summary of the neutralisation titres for sera raised against the recombinant viruses in mice (CSN-1 and CSN-2, by IC inoculation of 0.04ml inactivated virus) against homologous viruses and RV61, compared with equivalent titres for mice inoculated with inactivated wild type viruses (M9 = mouse inoculated with RV9 and M1787 = mouse inoculated with RV1787).

RV9 RV1787 RV61CSN1 (SADB19 backbone EBLV-1 G) 4096 362 6561M9 (Wildtype EBLV-1) 5793 2048 11634CSN2 (SADB19 backbone EBLV-2 G) 23 23 16M1787 (Wildtype EBLV-2) 45 45 47

Table 6 Neutralisation titres for sera raised against the recombinant viruses in mice

Analysis shows that sera from the mice inoculated with the recombinants have largely similar responses to sera from mice inoculated with the wildtypes. In general, the anti-wild type sera demonstrated higher titres.

04 Characterise newly identified Ikoma Virus


All milestones met

We have recently identified a novel lyssavirus species when screening infected material from a rabid African Civet (Tanzania). Using the resources released by the removal of milestones M03/03 and M01/04, we were able to further analyse this new lyssavirus.

M04/01 Submit a case report on the detection of a novel lyssavirus in an African Civet

On May 11, 2009, an African civet (Civettictis civetta) displaying clinical signs consistent with rabies was killed by rangers in Ikoma Ward within Serengeti National Park. Rangers were contacted because the civet had bitten a child on the right leg in an unprovoked attack. The wound was washed with soap and water, and the child received postexposure rabies vaccination but no rabies immunoglobulin. Brain samples from the civet were tested multiple times (as part of a training course) at the Central Veterinary Laboratory in Tanzania. Results of the fluorescent antibody test and a direct rapid immunohistochemistry test were positive for lyssavirus-specific antigen. When testing was complete, the samples were sent to the Animal Health and Veterinary Laboratories Agency (AHVLA, Weybridge, UK) for molecular analysis. Results of the phylogenetic analysis of partial nucleoprotein gene sequences from representative Lyssavirus species provided strong empirical evidence that the civet isolate represented a new lyssavirus species, designated Ikoma lyssavirus. The report was published in EID (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3309678/)

M04/02 Design primers and obtain N and G gene sequence data for the novel lyssavirus – met

Using NGS technology (454 sequencing), we have successfully obtained the full genome sequence for IKOV. Phylogenetic analysis of the full genome IKOV sequence with representatives from all lyssavirus species has been undertaken to investigate the relatedness of the lyssavirus genus. Table 7 summarises the gene and intergenic lengths of IKOV compared to other lyssaviruses. Figure 24 depicts a Maximum Likelihood analysis based on the Kimura 2-parameter model. Neighbour Joining analysis was also undertaken, the results were comparable to the ML analysis. The genome sequence was published in Journal of Virology http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3446578/

RABV WCBV IKOV All species

3' UTR 70 70 70?70? 7070

N protein 1353 1353 13531353 1353 or 13561353 or 1356N–P 90-1 64 66 64-102

P protein 894 894 870870 894-918894-918P–M 88 133 7474 72-13372-133

M protein 609 609 609 609M–G 211-5 206 209209 191-215191-215

G protein 1575 1578 1575 1569-1602G–L 522 862 569569 504-862504-862

L protein 6384 6384 6381 63845' UTR 131 125 126? 125-150

Genome 11 923-8 12278 11902 11902-12178




Table 7 Gene and intergenic lengths of RABV, WCBV and IKOV


Project SummaryThis project was highly successful in developing and applying molecular and serological tools to study the humoral responses and pathogenicity of emerging lyssaviruses. The data generated in this project has led to several publications. The pseudotype assays will have widespread applicability for a range of serosurveillance studies – including those proposed for BBLV. A multiplex system for EBLV-1, EBLV-2 and BBLV (employing three reporters) would be highly beneficial to those involved in bat surveillance in Europe.

The multiplex PNAs for African Lyssaviruses (LBV, MOKV and the newly identified Ikoma lyssavirus) could be utilised to survey bats and other species in further African countries. The PNA for WCBV could be used in Eurasian bat surveillance schemes where for biosafety reasons the virus cannot be handled. The IKOV PT would equally prove beneficial for further analysis of this novel virus, which may be assumed to have little or no protection from the classical vaccine strains due to its significant divergence from RABV. Antigenic cartography will be applied to IKOV to determine the efficacy of vaccine strains (under SE0427).

The SAD B19 reverse genetics component of the project in combination with site directed mutagenesis and antigenic cartography will provide a platform to further understand the genetic elements responsible for neuroinvasiveness and cell tropism. The promising validation data may further promote the use of the ptCVS-11 PNA for post vaccination serology in place of the live virus based assays (FAVN). Antigenic cartography will be employed to assess the vaccine and rabies immunoglobulin (RIG) efficacy against the new and emerging lyssaviruses (including IKOV and BBLV). In addition it will be used to assess candidate virus neutralising agents developed to replace RIG, such as monoclonal antibody cocktails or aptamers.


References to published material9. This section should be used to record links (hypertext links where possible) or references to other

published material generated by, or relating to this project.Horton DL, McElhinney LM, Marston DA, Wood JL, Russell CA, Lewis N, Kuzmin IV, Fouchier RA,

Osterhaus AD, Fooks AR, Smith DJ. Quantifying antigenic relationships among the lyssaviruses. J Virol.

2010 Nov;84(22):11841-8.

Fooks AR, Johnson N, Freuling CM, Wakeley PR, Banyard AC, McElhinney LM, Marston DA, Dastjerdi

A, Wright E, Weiss RA, Müller T. Emerging technologies for the detection of rabies virus: challenges

and hopes in the 21st century. PLoS Negl Trop Dis. 2009 Sep 29;3(9):e530

Marston DA, McElhinney LM, Banyard AC, Horton DL, Núñez A, Koser ML, Schnell MJ, Fooks AR.

Interspecies protein substitution to investigate the role of the lyssavirus glycoprotein. J Gen Virol. 2013

Feb;94(Pt 2):284-92. doi: 10.1099/vir.0.048827-0. Epub 2012 Oct 24.

Marston DA, Ellis RJ, Horton DL, Kuzmin IV, Wise EL, McElhinney LM, Banyard AC, Ngeleja C, Keyyu

J, Cleaveland S, Lembo T, Rupprecht CE, Fooks AR. Complete genome sequence of Ikoma lyssavirus.

J Virol. 2012 Sep;86(18):10242-3.

Marston DA, Horton DL, Ngeleja C, Hampson K, McElhinney LM, Banyard AC, Haydon D, Cleaveland

S, Rupprecht CE, Bigambo M, Fooks AR, Lembo T. Ikoma lyssavirus, highly divergent novel lyssavirus

in an African civet. Emerg Infect Dis. 2012 Apr;18(4):664-7. doi: 10.3201/eid1804.111553.

Wright E, Temperton NJ, Marston DA, McElhinney LM, Fooks AR, Weiss RA. Investigating antibody

neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison. J Gen Virol.

2008 Sep;89(Pt 9):2204-13.

Wright E, McNabb S, Goddard T, Horton DL, Lembo T, Nel LH, Weiss RA, Cleaveland S, Fooks AR. A

robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and lyssaviruses

Wright E, Hayman DT, Vaughan A, Temperton NJ, Wood JL, Cunningham AA, Suu-Ire R, Weiss RA,

Fooks AR. Virus neutralising activity of African fruit bat (Eidolon helvum) sera against emerging

lyssaviruses. Virology. 2010 Dec 20;408(2):183-9. Epub 2010 Oct 15.

Oral presentations

A robust lentiviral pseudotype neutralisation assay for in-field serosurveillance of rabies and


lyssaviruses in Africa. Oral presentation, Rabies in the Americas XX, Quebec, Canada, 18-22nd October

2010

Applications of antigenic cartography: Veterinary Training and Research Initiative Final Review Meeting,

Hinxton Nov 2009

Fooks, AR Development of a novel assay for detecting lyssavirus neutralising antibodies using lentiviral

pseudotypes, Epizone Brescia, Italy June 2009

Temperton, NJ. A Modular retroviral pseudotype framework for the evaluation of influenza virus,

lyssavirus and coronavirus neutralizing antibodies and inhibitors. SGM Annual Meeting Harrogate April

2009.

Quantifying antigenic relationships among the lyssaviruses. Oral presentation. SGM Spring Meeting,

Harrogate April 2009.

Wright, E., Armson, B., McElhinney, L.M., Banyard, A., Goddard, T., Healy, D., Voller, K., Weiss, R.,

Fooks, A.R. (2010) Development of a novel serological assay for the detection of rabies virus neutralising

antibodies using lentiviral pseudotypes. Poster Presentation. 4th Annual Meeting Epizone. 7-10 June

2010, St Malo, France

Healy et al (2013) Pseudotype neutralisation assay inter-laboratory trial (2013) Society general

microbiology, Manchester March 25th-28th

McElhinney, LM Development and Validation of lyssavirus Pseudotype Assays (2013) Community

Reference Lab Serology meeting, nancy France, October 1st -2nd


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