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Evidence Project Final Report

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 21

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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 SE0423

2. Project title

Molecular and Antigenic Evolution of Lyssaviruses

3. Contractororganisation(s)

Animal Health and Veterinary Laboratories AgencyWoodham Lane, New HawAddlestone SurreyKT15 3NB

54. Total Defra project costs £ 685,775(agreed fixed price)

5. Project: start date................ 01/04/2008

end date................. 31/03/2012


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 In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain.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.

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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.

The project had four main objectives:

01 Acquisition and analysis of RABV from rabies endemic areas, particularly Eastern Europe, Africa and Asia. [47 months]We have assembled an extensive Lyssavirus Reference Virus Archive composed of over 2500 lyssaviruses and the partial genetic sequence of over 2000 lyssaviruses. The utility of such a database grows in proportion to its representative size with respect to host species, geographical location, chronological isolation and lyssavirus species. During the course of this project, the AHVLA Lyssavirus Reference Virus Archive has been expanded by the acquisition of isolates responsible for new outbreaks and existing epizootics in various countries globally. In particular, we have obtained viruses or viral genetic material from the previously understudied areas of Eastern Europe, Asia and Africa. Over the 4 years we have received samples from 10 African, 5 Asian and 6 European countries and a number of other countries such as Mexico and Grenada. We have published 13 manuscripts, relating to viruses held within our archive, mainly utilising phylogenetic analyses on partial N gene sequences investigating both geographical and host species interactions. In addition, during this project, we have implemented the use of evolutionary clock analysis, requiring full N and G gene data which enables more detailed analyses of extensive panels such as a panel of Balkan viruses, collected over thirty years from a range of host species in geographically distant regions of the former Yugoslavia. The importance of continuing the

02 Expand and consolidate the lyssavirus antigenic map to quantify the antigenic differences among the most globally divergent lyssaviruses. [35 months]In collaboration with CDC, FLI and Cambridge University, antigenic cartography has been applied to the emerging lyssaviruses (Irkut, Khujand, West Caucasian Bat and Aravan viruses), and representatives of African lyssaviruses. A publication detailing the significant findings has been published during this project, along with the successful completion of a PhD by a student in collaboration with the Cambridge Infectious Diseases Consortium. We have described the antigenic relationships among a panel of 25 lyssaviruses using serological binding assay data and antigenic cartography. This approach has quantified clinically important antigenic differences between lyssaviruses; shown that those differences are equivalent for mouse, rabbit, and pooled human sera; and allowed integration of quantitative antigenic data with genetic distances. We have investigated the utilization of the cartography software to analyse amino acid sequences from the full glycoprotein sequence of the panel of Balkan viruses, collected over thirty years from a range of host species in geographically distant regions. Partial glycoprotein ectodomain sequences


for twenty five representative viruses from the Balkans, and eight control viruses were included in the investigation. There is no obvious pattern of antigenic evolution (as measured by glycoprotein identity) over time, with viruses from the 1970s, 1980s and 1990s clustering together. All viruses on the map isolated after 2000 are, however, in one region of the map suggesting a possible development of an antigenically distinct cluster. Comparison of glycoprotein similarity with isolation host gives a similar lack of distinct pattern. Finally, comparison of amino acid similarity with the region show the viruses from Central/Southern Serbia, and Montenegro are spread throughout the map with only the isolates from Bosnia Herzegovina grouping closer to each other than any other viruses.

03 To investigate quasispecies in EBLV-2 by performing in vitro and in vivo serial passages. [24 months]Quasispecies, representing nucleotide sequence heterogeneity of the viral RNA, were originally proposed for rabies virus as a mechanism for viral evolution and adaptation to a new host facilitating animal to animal transmission and possible maintenance in a new reservoir species. In this project we have obtained the full genome sequence for an EBLV-2 bat isolate and compared it to a previously obtained human EBLV-2 additionally we have investigated both in vitro and in vivo passaged material and identified regions where polymorphisms were observed. During the project, AHVLA as acquired both an Ilumina and 454 Next Generation Sequencers (NGS). We have successfully developed two full genome sequencing methodologies: 1) Consensus Sequencing and 2) amplicon ‘deep’ sequencing. The consensus sequencing method is applied to obtain sequence an unknown virus (or virus for which full genome sequence is not currently available). RNA is extracted from original brain material or tissue culture supernatant without using primers. We have had exceptional success using the consensus sequence methodologies with 11 full length genomes (10 Lyssaviruses) obtained by the end of this project. The deep sequencing method utilises overlapping PCR amplicons designed against a known virus genome sequence to ‘deep sequence’ a virus genome to investigate quasispecies. This methodology requires the consensus sequence to be available, either by using the method above or published full genome sequences in genbank. Using the complete genome sequence obtained from our EBLV-2 isolates we have designed primers and have obtained overlapping amplicons for the original brain material of two EBLV-2 isolates. These amplicons have been sequenced on the NGS platform. The sequencing reads obtained for each amplicon provide depth of coverage to enable analysis of minority viral populations.

04 To improve upon the VLA Lyssavirus Reference Virus Archive [26 months]There were two systems which were in need of improvement with respect to the AHVLA Lyssavirus Ref Lab. The first relates to the logistical tracking of the archived material (virus, RNA, cDNA and associated biproducts) whilst the second relates to the storage of sample data (submission, clinical history, test data, sequence data). Both these improvements were achieved by the implementation of ICAST (Inventory control and sample tracking) an SQL database designed in partnership with the HPA and ECACC, to enable quick data entry and tracking of millions of samples. We have successfully trialled, reviewed and improved ICAST over a number of years to tailor the program to suit our needs. It is now installed on our server and is being used to catalogue and trace our lyssavirus archive. In addition, in collaboration with National Centre for Zoonosis Research (NCZR) we have developed an online sequence database to enable easy searches of all available lyssavirus sequences with downloadable outputs. This database interfaces with genbank, constantly updating to ensure the latest lyssavirus sequences are captured. The search facilities and features built into this database make it an extremely useful too. The rabies virus database is live and is used by ourselves and a number of collaborators.

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 objectives and milestones have been fully met.

01 Acquisition and analysis of RABV from rabies endemic areas, particularly Eastern Europe, Africa and Asia. [33 months]

Milestones for this objective:M01-01 Submit a report on the molecular epidemiology of Rabies viruses in Sudan (31/08/2008)M01-02 Submit a report on the phylogenetic analysis of Serbian RABV (31/10/2008)M01-03 Submit a report on the molecular epidemiology of Rabies viruses in Namibia (31/08/2009)M01-04 Submit a report on the molecular epidemiology of Rabies viruses in Eurasia (31/12/2010)M01-05 Submit a report on the molecular epidemiology of Rabies viruses in Africa (29/02/2012)

Table 1 Viruses submitted to AHVLA for Genetic Sequencing (2008-11)

Country Viruses Submitted

Spain 1 Spanish bat isolate (EBLV-1)

Germany 1 German bat isolate (EBLV-1)2 German bat isolates (EBLV-2)1 German Natter’s bat isolate (BBLV)1 Pasteur Virus (RABV)

UK (Ex Sri Lanka) 1 Dog isolate in England Quarantine (Ex Sri Lanka)

UK (Ex S.Africa) 1 Human RABV isolate from England (ex S Africa)

UK 2 UK bat isolates (EBLV-2)

Nigeria 53 RABV dog isolates from Nigeria

Ethiopia 4 RABV isolates from Ethiopian Wolves

Switzerland 3 Swiss bat isolates (EBLV-2)

Tanzania 249 X brain samples from Tanzania

Ethiopia 6 x Brain samples +other organs - Ethiopian Wolves

Ghana 96 x Dog Brain samples from Ghana

Germany 10 x Fox Brain or head samplesSudan 126 x Brain samples from a variety of speciesTanzania 55 x brain material matching FTA card samples for

comparisonMorocco 246 x brain samples from a variety of speciesIraq 40 x brain samples from dogs Grenada 35 x mongoose, dog, cat, goat and opossum brain samplesSri Lanka 32 x brain samples from DogsSaudi Arabia 3 x animal brain samples

Table 2 Viral RNA/inactivated virus FTA submitted to AHVLA for Genetic Sequencing (2008-11)

Country Viral RNA Submitted

Spain 1 x RNA EBLV-1


Germany 1 x RNA EBLV-126 African RABV RNAs (Tunisia, Egypt, Ethiopia, Algeria and Sudan)1 x MOKV RNA6 x RABV Samples from 3 foxes (Poland, Russia and Germany), 1 dog (Sudan), 2 humans (Chile)

Turkey 10 x FTA cards, 1 cat, 2 cows and 7 dogs

Mexico 9 x FTA cards, 1 cat, 4 cows, 2 dogs, 1 skunk, 1 horseIndia 64 x FTA cards (containing inactivated dog brain samples)

Tanzania 55 x FTA cards containing brain material from FAT confirmed cases

Sudan 12 x FTA cards from bat brains

UK EBLV-2 RNA positive bat saliva (active surveillance)

Over the course of this project we have continued to obtain sequence data (400bp) at the 5’ end of the nucleoprotein gene from a large number of the virus isolates in our archive. In addition, full N and G gene data has been obtained for an increasing number of isolates. Phylogenetic studies on the N gene sequences continue to demonstrate numerous groups of lyssaviruses clustered at both geographical and host species levels (published and unpublished data presented at several national and international conferences).

During this project we have acquired two novel lyssaviruses: Bokeloh bat lyssavirus (BBLV) isolated in Germany from a Natterer’s bat in 2010. Preliminary analysis on

this virus indicates it is a new phylogroup 1 lyssavirus most closely related to KHUV and EBLV-2. We received this virus from our German collaborators in 2011 and have been working in collaboration to help characterise the virus further and determine the efficacy of our diagnostic tests (SE0536).

Ikoma Lyssavirus (IKOV) isolated at AHVLA from an African civet sample sent from Tanzania in 2011. This sample was sent with a cohort of lyssavirus positive samples (tested by FAT) and was expected to be a RABV. By sequencing the 400bp N-gene PCR product we were able to determine that this was in fact a novel lyssavirus. Moreover, this lyssavirus is the most diverse lyssavirus documented. We have isolated virus and have fully sequence the genome using 454 sequencing technologies. Samples from this case have been sent to CDC-USA as part of an ongoing collaboration.

Achievements from this objective, listed by Milestone then country/region set out in objectives.

Milestone completion:

RABV in Sudan : Publication Marston et al 2008 (Milestone M01/01)Rabies is endemic in Sudan and remains a continual threat to public health as transmission to humans is principally dog-mediated. Additionally, large-scale losses of livestock occur each year causing economic and social dilemmas. In this study, we analysed a cohort of 143 classical rabies virus (RABV) strains circulating in Sudan collected from ten different animal species between 1992 and 2006. Partial nucleoprotein sequence data (400bps) were obtained and compared to available sequence data of African RABV isolates. The Sudanese sequences formed a discrete cluster within the Africa 1a group, including a small number of sequences that clustered with sequences from Ethiopian strains. These latter sequences share an Aspartic Acid at position 106 (Asp106) with all other Africa 1a group members, in contrast to the remaining Sudanese strains, which contain Glutamic Acid at this position (Glu106). Furthermore, when representatives of other African and European lineages were aligned, Glu106 is unique to Sudan, which supports the concept of a single distinct virus strain circulating in Sudan. The high sequence identity in all Sudanese isolates studied, demonstrates the presence of a single rabies virus biotype for which the principal reservoir is the domestic dog. Molecular diversity and evolutionary history of rabies virus strains circulating in the Balkans: Publication McElhinney et al 2011 (Milestone M01-02)Studies on the molecular diversity of RABV in Europe have revealed a number of geographically clustered lineages. To investigate RABV in The Balkans, a molecular epidemiological study (Nucleoprotein (N) gene, 400bp) was undertaken on a unique panel of viruses (n=210), collected in the region from a wide range of hosts


over a 34-year period. All of the Balkan isolates grouped within the European / Middle East Lineage, the majority of which were most closely related to the previously published East European (EE) strains. A number RABV isolates from Bosnia & Herzegovina (BiH) and Montenegro, collected between 1986 and 2006, grouped with the West European (WE) strains, believed to be responsible for the rabies epizootic that spread throughout Europe in the latter half of the 20th Century. In contrast, no Serbian RABV isolates in this study belonged to this sub-lineage. However, a distinct group of Serbian fox rabies isolates provides further evidence for the southwards wildlife-mediated movement of rabies from Hungary, Romania and Serbia into Bulgaria. To investigate the optimal region for evolutionary analysis, partial, full and concatenated N-gene and Glycoprotein (G) gene sequences were compared for a subset of the Balkan viruses. Whilst both the divergence times and evolutionary rates are similar irrespective of genomic region, the 95% highest probability density (HPD) limits are significantly reduced for full N-gene and concatenated NG-gene sequences compared to partial N-gene and G-gene sequences. Bayesian coalescent analysis estimated the date of the most common recent ancestor of the Balkan RABV to be 1885 [95% HPD,1852–1913], and skyline plots suggested an expansion of the local viral population in 1980-1990 which coincides with the observed emergence of fox rabies in the region.

Rabies disease dynamics in Namibia: Publication in prep (Milestone M01/03)Our previous molecular analysis of rabies isolates from Namibia suggested that the domestic and sylvatic cycles interact closely, and that rabies is a transboundary disease; viral strains isolated from neighbouring countries Botswana and South Africa were closely related to strains within Namibia [Mansfield 2004]. Through continued collaboration with the Namibian Central Veterinary laboratory, Windhoek, we have used an expanded panel of more recent isolates to further investigate the molecular epidemiology of rabies in Namibia.

The cohort of 123 isolates included 45 cases originating from the northern communal areas of Namibia, the most densely populated region. Six samples were from Etosha National Park, the majority were from central cattle ranching areas and only four were from the southern sheep rearing areas. In total 14 species were represented, the main groups being kudu antelope (n=35), dog (n=28) and jackal (n=16). The panel has also isolates originating from cow, goat, sheep, pig, cat, bat eared fox, honey badger, hyena, eland, horse and slender mongoose. The 37 previously published RABV nucleotide sequences from Namibia, and representative isolates from elsewhere in Africa were also included in the analyses.

Neighbour joining and Bayesian coalescent analyses were carried out on a 400bp region of the nucleoprotein gene using Megalign (DNAStar) and BEAST v1.4.8 respectively. The neighbour-joining tree was constructed with only unique sequences (n=36), Bayesian analyses were carried out on all sequences.

Our analysis confirms that all the isolates belong to 1b Africa group. More detailed analysis shows the tree has four distinct groups. Group 1 includes mainly kudu isolates from the cattle ranching areas in central and west Namibia. The isolates are characterised by a serine at N aa position 36. The second group (group 2, 22 isolates) is defined by a leucine at the amino acid position 36. It contains nearly exclusively samples from the Northern communal areas except one Kudu and one cow from central areas. The biggest group of isolates within this group originate from the North Central Division of the northern communal areas.

These data show the very close genetic relationships between isolates from multiple different species and different regions of Namibia. Although there is some clustering of isolates according to geographic region, isolates that are genetically identical (over the nucleoprotein region studied), have been detected in multiple species, and over large geographic areas. This provides challenges for control of disease, showing that eradication of disease in one reservoir such as domestic dogs will not stop the disease circulating.

The majority of kudu isolates are closely genetically related. A relaxed molecular clock model implemented with the Bayesian MCMC algorithm in BEAST, suggests that the most recent common ancestor of the isolates found in kudu occurred 26-36 years ago, which corresponds to historical accounts of the emergence of rabies in Kudu. However, there are isolates from other known reservoirs within that clade, including dog and jackal. Therefore there is insufficient evidence from these molecular data that kudu are able to maintain infection without other reservoirs.

A report has been sent to collaborators, but work is currently underway to investigate quantitatively the temporospatial dynamics of the disease, and relative rates of evolution among isolates from each area and species. These more advanced analyses will be included in a manuscript currently being prepared for publication.

Molecular Epidemiology of lyssaviruses in Eurasia: Publication McElhinney et al 2008 (Milestone M01-04)Rabies cases in Eurasia are principally attributed to three lyssavirus species, mainly RABV (classical rabies) and to a lesser extent EBLV-1 and -2 (European bat lyssaviruses type-1 and -2). In addition, four newly identified divergent lyssaviruses have been isolated from insectivorous bats. The molecular diversity of RABV has been studied at the global level and reference has been made to the existence of a number of European strains in a range of mammalian species. It is accepted that these viruses cluster within a 'Cosmopolitan Lineage' having


ancestral roots in Europe in the 17th century before its widespread dispersal to Asia, Africa and the Americas as a result of European exploration and colonisation.

Molecular Diversity of Rabies Virus Strains In Nigeria: Publication in prep Milestone M01/05In 2009, we received and analysed a cohort of 140 FAT positive dog samples from the Plateau State covering rural and city areas (n=123) and other states (n=17) in Nigeria, sampled between 2004 and 2008. From this cohort, 128 were positive by hnRT-PCR using nucleoprotein specific pan-lyssavirus primers. Phylogenetic analysis revealed that only RABV (Genotype 1) was present. Further analysis with representative sequences from other African countries, aligned the Nigerian sequences in the Africa 2 lineage, which is restricted to Northern Africa and endemic within the domestic dog.

In collaboration with Dr Dan David (Kimron Veterinary Institute, Israel and Ahmadu Bello University of Zaria, Nigeria), a further twenty-four canine RABV samples were collected from the Plateau State, Nigeria and complete N gene sequences (N1350) were obtained. Previous phylogenetic analysis of Africa 2 isolates (Talbi et al 2008) revealed 8 sub-lineages (Groups A-H). The Nigerian isolates for which full N gene data were available in this study were present in only Groups D and E of Africa 2. In addition, a previously published human isolate from Nigeria (U22488 1986) remained unique as an Africa 1b isolate. Using partial N gene sequences, Nigerian RABV were resolved in three Africa 2 sub-lineages possibly representing three separate incursions into Nigeria from Benin (Group D), Chad (Group E) and Niger/Burkina Faso (Group F). The majority of the Nigerian RABV were Group E viruses.

Figure 1. MCC tree of 175 partial nucleoprotein sequences (400 nucleotides) derived from RABV from Nigeria and bordering African countries. The estimated TMRCA for this sample of viral lineages are indicated. The major groups (A–H) of the Africa 2 clade are also indicated.

Previously published partial N gene sequences (N400) originating from Nigeria (including n=19 Talbi et al 2008, n= 54 Ogo et al., 2008) were combined with those available at AHVLA and with published sequences from neighbouring countries in sub-Saharan Africa. Identical sequences were removed for ease of analysis and the remaining dataset consisted of 175 sequences. Molecular clock analysis was performed on the 2 data sets (N400 and N1350). The maximum clade credibility (MCC) phylogenetic tree for the larger panel (n=175) of partial N gene (400) sequences is shown (Fig 1). Molecular clock analysis inferred that the time to the most recent common ancestor for the Nigerian isolates in the Africa 2 Group D was 23 years (1985) and these viruses share a common ancestor with an isolate from Benin and a group of viruses from Ghana. This is the first report of


30.0

Ghana 2007-2008

EU853636N_Sen_Human_2006

EU853591N_Gam_Dog_2008

EU478515N_BF_2007

EU853613N_Maur_Ass_1991

U22488N_Nigeria_1986

U22628N_AFS_Mong_1987

EU853582N_Rwa_Dog_1995

EU853568N_Alg_Human_1996

U22638N_DRC_Dog_1989

EU853653N_Chad_1996

EU853614N_BF_1986

EU038107N_Nigeria_2006

U22627N_Egy_Human_1979

EU853595N_SL_Cat_1997

Chad 2005-2006

EU853566N_Tun_Human_1986

Nigeria 2002-2008

AY103008N_Nigeria_Human_1996 EU718771N_Chad_200

6

EU853652N_Chad_1990

EU853657N_Chad_1987

U22649N_Nam_Jackal_1992

U22650N_CAR_Dog_1992

EU853602N_Mali_Dog_2006


U22485N_Benin_1986

EU853569N_Mor_Dog_1990

EU478502N_BF_2007

EU853583N_Bur_Jackal_1990

EU478518N_BF_2007

U22644N_Chad_1992

99N_Nigeria_2005

EU853592N_Sen_Human_2005

GhanaHM368121_2008

DQ420623N_Mad_Human_2004

EU718783N_Chad_2006

U22863N_Niger_1987

EU853587N_CAR_Dog_2006

EU853581N_Maur_Goat_2007

U22484N_Moz_Fox_1986

EU853651N_CAR_Dog_2004

EU853594N_Gui_Dog_1993

EU853579N_Tun_Human_1986

U22645N_Tan_Dog_1992

EU853622N_Mali_Dog_2006

GhanaHM368094_2008

EU718736N_Chad_2005

EU853615N_IC_Dog_2007

U22636N_Cam_1988

U22634N_Cam_1987

161N_Nigeria_2005

U22635N_Cam_1988 EU718765N_Chad_200

6

GhanaHM368142_2008


RV2369N_Nigeria_2006

EU478492N_BF_2007

2000

1933

1915

1993

1835

1974

1971

1983

1990

1934

1991

1885

1935

1906

1985

1964

1984

1719

1783

1892

1947

1960

1944

1915

Ghana 2008

Nigeria 2004-2007

Ghana 2008

Burkina Faso 2007

Niger 2007

Niger 1990Burkina Faso

1995Nigeria 2007

Nigeria 1996-2007

U22488 Nigeria 1986

AFRICA 4

AFRICA 1

AFRICA 2

AFRICA 3

Group D

Group E

Group HGroup G

Group F

Group A

Nigeria 2005

Group C

Group B

Nigerian RABV in the Group D sub-lineage. The Group E viruses, representing the majority of the Nigerian RABV analysed, share a common ancestor with viruses from Chad and Cameroon which diverged around 1960. The Group F Nigerian RABV share a common ancestor with viruses from Niger, Burkina Faso and Ghana which diverged around 1964. The TMRCA for the Africa 2 lineage is estimated to be 1892 (later than estimated by Talbi probably due to the inclusion of a greater number of sequences and the shorter genomic region). A report has been prepared and circulated to collaborators prior to publication.

Lyssaviruses from Europe:

EBLV Cases in Germany: Publication Freuling et al 2008Germany has reported one of the highest levels of EBLV cases in bats in Europe. Prior to 2008, all isolates originating from Germany were identified as EBLV-1, using monoclonal antibodies, and a preliminary epidemiological study has indicated that there is a distinct geographical distribution of EBLV-1 in Germany. To further investigate the spatial and temporal distribution of EBLV-1 variants in Germany and their impact on molecular epidemiology, isolates were selected using a random grid sampling procedure based on GIS. A grid layer 30 km long over the entire area of Germany was applied to 120 geo-referenced isolates and one isolate of each grid cell containing EBLV isolates was randomly chosen. Once selected, the nucleoprotein (N) plus parts of the phosphoprotein (P) gene of each isolate were sequenced using direct cycle sequencing. Results of the subsequent phylogenetic analysis of the N-gene confirmed previous studies on EBLVs, showing a high sequence homology between German EBLV-1 isolates. Almost identical sequence homologies within certain geographical regions indicate genomic stability during the transmission cycle of EBLV-1, with little geographic spread or intermixing. Interestingly, a 6 bp insertion as well as a single nucleotide insertion, detected in the N-P intergenic region, has been found in EBLV-1 isolates from Germany.

RABV in Southeast Europe: Publication Johnson et al 2008Rabies remains endemic within a number of countries in Southeast Europe including Romania, Bulgaria and Turkey. With the probable expansion of the European Union eastwards, it is likely that rabies elimination programs will be increased to reduce the burden of disease in new accession countries. A clear understanding of the epidemiology of the virus in this area of Europe is vital before such programs are introduced. With the exception of Turkey, the red fox is the principal disease reservoir in Southeastern Europe. However, cases of rabies in the dog are regularly reported. In contrast to Northern Europe, the raccoon dog does not appear to be a vector in the south. Rabies virus RNA was extracted from original samples and a fragment of the nucleoprotein gene amplified by RT- PCR. Nucleoprotein gene sequences were used to prepare a molecular phylogeny of rabies viruses in Southeast Europe. In Bulgaria, the dog is the main vector bringing rabies into contact with humans and livestock. However, other species may also act as reservoirs for the disease, complicating the development of elimination strategies. The fox is the principal reservoir species for rabies in Romania although cases in dogs are regularly reported. Despite a gradual decline in dog rabies, urban pockets of the disease remain in many regions of Turkey. Furthermore, there is some evidence that the fox has been a significant vector for RABV and may be responsible for increases in rabies in cattle in the Aegean region of the country. Throughout the region there is evidence for cross-border movement of rabies by both wildlife and canine vectors. Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife: Publication Geue et al 2008The elimination of rabies from the red fox (Vulpes vulpes) in Western Europe has been achieved by the oral rabies vaccination (ORV) of wildlife with a range of attenuated rabies virus strains. With the exception of the vaccinia rabies glycoprotein recombinant vaccine (VRG), all strains were originally derived from a common ancestor; the Street Alabama Dufferin (SAD) field strain. However, after more than 30 years of ORV it is still not possible to distinguish these vaccine strains and there is little information on the genetic basis for their attenuation. We therefore sequenced and compared the full-length genome of five commercially available SAD vaccine viruses (SAD B19, SAD P5/88, SAG2, SAD VA1 and SAD Bern) and four other SAD strains (the original SAD Bern, SAD VA1, ERA and SAD 1-3670 Wistar). Nucleotide sequencing allowed identifying each vaccine strain unambiguously. Phylogenetic analysis revealed that the majority of the currently used commercial attenuated rabies virus vaccines appear to be derived from SAD B19 rather than from SAD Bern. One commercially available vaccine virus did not contain the SAD strain mentioned in the product information of the producer. Two SAD vaccine strains appeared to consist of mixed genomic sequences. Furthermore, in-del events targeting A-rich sequences (in positive strand) within the 3' non-coding regions of M and G genes were observed in SAD-derivates developed in Europe. Our data also supports the idea of a possible recombination that had occurred during the derivation of the European branch of SAD viruses. If confirmed, this recombination event would be the first one reported among RABV vaccine strains.

Molecular Epidemiology of Bat Lyssaviruses in Europe: Publication McElhinney 2012 in press


Bat rabies cases in Europe are principally attributed to two lyssaviruses, namely European Bat Lyssaviruses type 1 (EBLV-1) and European Bat Lyssavirus type 2 (EBLV-2). Between 1977 and 2011, 958 cases of bat rabies were reported to Rabies Bulletin Europe, with the vast majority (>97%) being attributed to EBLV-1. To date, there have been 25 suspected cases of EBLV-2, of which 22 have been confirmed. In addition, two single isolations of unique lyssaviruses from European insectivorous bats were reported in South West Russia in 2002 (West Caucasian Bat Virus) and in Germany in 2009 (Bokeloh Bat lyssavirus). In this review, we present phylogenetic analyses of the EBLV-1 and -2 using partial nucleoprotein (N) gene sequences. In particular, we have analysed all EBLV-2 cases for which viral sequences (N gene, 400 nucleotides) are available (n=21). Oro-pharyngeal swabs collected from two healthy Myotis Daubentonii during active surveillance programmes in Scotland and Switzerland also yielded viral RNA (EBLV-2). Despite the relatively low number of EBLV-2 cases, a surprisingly large amount of anomalous data have been published in the scientific literature and Genbank, which we have collated and clarified. For both viruses, geographical relationships are clearly defined on the phylogenetic analysis. Whilst there is no clear chronological clustering for either virus, there is some evidence for host specific relationships, particularly for EBLV-1 where more host variation has been observed. Further genomic regions must be studied, in particular for EBLV-1 isolates from Spain and the EBLV-2 isolates to provide support for the existence of sub-lineages.

Lyssaviruses from Africa:

IKOV: a novel Lyssavirus isolated from an African Civet: Publication Marston et al 2012On 11th May 2009 an African civet displaying clinical signs consistent with rabies, was killed by rangers in the Ikoma ward, within the SNP. 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 rabies post-exposure vaccination, but no rabies immune globulin. Viral antigen was detected by the fluorescent antibody test (FAT) on civet brain smears at the Central Veterinary Laboratory, Tanzania. Viral RNA was extracted using TRIzol® and a pan-lyssavirus RT-PCR yielded a specific 606-bp amplicon which was sequenced and compared with representatives from all species of the lyssavirus genus using Bayesian reconstructions. These data showed that the sequence was unique and most closely related to WCBV. Both dog RABV from Tanzania and a mongoose RABV biotype from Southern Africa were included in the dataset. Nucleotide similarity comparisons indicated similar divergence from all lyssavirus species (minimum identity 62.2% ABLV - maximum identity 68.6% WCBV), including 12 canine RABV sequences from domestic and wild animals in the Serengeti ecosystem (64.1% - 65.1%). The posterior probabilities indicated that the IKOV and WCBV grouping was strongly supported, despite the low sequence identity. Further phylogenetic analysis using representatives from other rhabdoviruses, demonstrated that IKOV is a member of the lyssavirus genus and the nucleotide identity to representative rhabdovirus sequences available for this region of the genome ranged from 41.6%-50.9%. This case was highly unexpected, located in the centre of the SNP, which had been rabies-free for over 8 years, with no detected cases within a 30km radius. The appearance of a rabies case in wildlife had implied a major breach in the vaccination program. Subsequent molecular characterization clearly demonstrated that this rabies case was not a RABV from a canine source and therefore no breach had occurred, but was instead a novel lyssavirus for which the reservoir is unclear. Since the publication we have isolated the virus and propagated it both in vitro and in vivo.

Arican Bat Lyssaviruses: Publication Markotter et al 2008Lyssaviruses belonging to all four known African Lyssavirus species have been reported and isolated from South Africa over the past few decades. These are: (1) Duvenhage virus (DUVV), isolated again in 2006 from a human fatality; (2) Mokola virus (MOKV), isolated irregularly, mostly from cats; (3) Lagos bat virus (LBV) continually isolated over the past four years from Epomophorus fruit bats and from incidental terrestrial animals and (4) Rabies virus (RABV) - with two virus biotypes endemic in mongoose and in canid species (mostly domestic dogs, jackals and bat-eared foxes), respectively. Only two of these are associated with bats in Southern Africa, i.e. DUVV and LBV. For both these species we have embarked on a programme of comparative study of molecular epidemiology. DUVV nucleoprotein nucleotide sequence analysis indicated a very low nucleotide diversity even though isolates were isolated decades apart. In contrast, individual isolates of LBV were found to differ significantly with respect to nucleoprotein gene nucleotide sequence diversity as well as in pathogenicity profiles.

Evolutionary history of rabies in Ghana: Publication Hayman et al 2011We confirmed the presence of RABV in a cohort of 76 brain samples obtained from rabid animals in Ghana collected over an eighteen-month period (2007-2009). Phylogenetic analysis of the sequences obtained confirmed all viruses to be RABV, belonging to lineages previously detected in sub-Saharan Africa. However, unlike earlier reported studies that suggested a single lineage (Africa 2) circulates in West Africa, we identified viruses belonging to the Africa 2 lineage and both Africa 1a and 1b sub-lineages. Phylogeographic Bayesian Markov chain Monte Carlo analysis of a 405bp fragment of the RABV nucleoprotein gene from the 76 new sequences derived from Ghanaian animals suggest that within the Africa 2 lineage three clades co-circulate with


their origins in other West African countries. Africa 1a is probably a western extension of a clade circulating in central Africa and the Africa 1b virus a probable recent introduction from eastern Africa. We also developed and tested a novel reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assay for the detection of RABV in African laboratories. This RT-LAMP was shown to detect both Africa 1 and 2 viruses, including its adaptation to a lateral flow device format for product visualisation. A new outbreak of rabies in rare Ethiopian wolves: Publication Johnson et al 2010Between October, 2008, and May, 2009, five brain samples from the carcasses of the rare Ethiopian wolf (Canis simenensis) were submitted for rabies virus testing. RABV was detected in all five samples and confirmed that a further outbreak of rabies had occurred within the wolf population within the Bale Mountains, Ethiopia. Sequence comparison of a partial fragment of the nucleoprotein coding gene demonstrated that all viruses showed 100% sequence identity suggesting a single introduction of rabies virus. Phylogenetic analysis demonstrates that a virus detected in the May 2009 sample is identical to those reported in October 2008 samples. The likely source of the October cases is the dog reservoir in and around Bale Mountains, although it is unclear whether the May 2009 case occurred due to wolf-to-wolf transmission from the earlier outbreak in Web Valley despite a vaccination intervention, or was a repeated spill-over from the dog population.

Mokola Virus (MOKV) evolutionary analysis: Publication in prepIn collaboration with the University of Pretoria (South Africa) and Friedrich Loeffler Institute (FLI, Germany), we are sequencing all available Mokola viruses. There are only a limited number of MOKV isolations and this work aims to provide the most comprehensive study of the MOKV isolates. In an attempt to identify and characterise what viruses have been submitted to the three virus archives over many decades, an inventory was taken at each institute and collated. Each institute was then tasked with obtaining partial genome sequence data for their viruses (N, P, M, and G gene data). Some of the older isolates had been submitted to all three labs and had been stored and passaged (in vivo and in vitro) for at least two decades. This study will facilitate a comprehensive intra-genotypic analysis of the Mokola viruses.

Molecular analysis of RABV from Tanzania – FTA card trial, publication in prepThrough a continued collaboration with Glasgow University and Central veterinary Laboratory in Tanzania, we have received 47 brain samples. In addition, duplicate samples were sent on FTA cards. All brain samples were tested by FAT in Tanzania and results recorded and sent to AHVLA. Brain homogenates for each sample were prepared put on to FTA cards just before time of sending, and then shipped to the AHVLA for testing along with original brain material. Of the 25 samples brain positive by FAT in Tanzania and that had corresponding FTA card samples, 22 were detected as RABV positive. A representative number of the FTA positive samples were sequencing to determine whether the product from an FTA card can be sequenced and phylogenetic analysis was undertaken. The results show that PCR products from FTA cards can be sequenced and that the sequences obtained group with previous isolates from Tanzania.

RABV in MoroccoA recently initiated collaboration within Morocco has resulting in us receiving 246 brain samples from a variety of species within Morocco. RNA has been extracted from these samples using the MELT RNA extraction kit and Kingfisher robot. Using real-time RT-PCR, only 2 samples were negative for rabies.N gene PCR and sequencing is in the process and phylogenetic analysis/BEAST analysis will be undertaken.

Lyssaviruses from Asia:

Rabies virus in a dog imported to the UK from Sri Lanka: Publication Fooks et al 2008We reported a case of classical rabies virus (RABV) in a 10-week-old dog imported from Sri Lanka to the UK. This case of rabies in an imported dog is the first in the UK since 1990. The dog was part of a single consignment of 13 animals that was imported to the UK by an animal charity involved with rescuing and re-homing stray ‘street’ dogs from Sri Lanka. Phylogenetic analysis of this consensus sequence confirmed that the virus was a wild-type strain of classical rabies virus of dog origin. The viral sequence aligned with other closely related canid isolates held in the AHVLA lyssavirus archive from Sri Lanka.

Characterisation of rabies virus from a human case in Nepal: Publication Pant et al 2011We report a case of rabies in a 12-year-old girl in the Lalitpur district of Nepal that might have been prevented by better public awareness and timely post-exposure prophylaxis. Molecular characterisation of the virus showed 100% identity over a partial nucleoprotein gene sequence to previous isolates from Nepal belonging to the ‘arctic-like’ lineage of rabies virus. Sequence analysis of both partial nucleoprotein and glycoprotein genes showed differences in consensus sequence after passage in vitro but not after passage in vivo.


India FTA cardsThe current legal regulations in India make exporting viral isolates, or positive material impossible. Therefore, there is only limited information regarding the rabies status and viral variants circulating in India. As a result of the successful validation work described above (Tanzania), we have supplied a collaborator in India with FTA cards and received 64 with brain smears from suspect street dogs. Only 1 positive was detected, real-time assays were also used to confirm these results because they utilise a smaller amplicon (165 bp) increasing the sensitivity, particularly when testing degraded samples, but no further positives were detected. The sequence from the positive sample grouped with Indian sequences published in Genbank.

Lyssaviruses from other regions:

Human rabies case with long incubation, Australia: Publication Johnson et al 2008We analysed the origin of a RABV sequence from 1990 human case of rabies in Australia. Phylogenetic analysis confirmed that the 10 year old girl had been exposed to an Asian strain of RABV, most likely originating from China. These findings exclude the possibility that the young Vietnamese girl was infected with ABLV, now known to be endemic within Australian bats, after she arrived in Australia.

A number of other related publications are listed in the publication section

02 Expand and consolidate the lyssavirus antigenic map to quantify the antigenic differences among the most globally divergent lyssaviruses. Publication Horton et al 2010 Milestone 02-03

Milestones for this objective:M02-01 Obtain antisera against and apply antigenic cartography to the African lyssaviruses (31/12/2008)M02-02 Obtain antisera against and apply antigenic cartography to the Emerging lyssaviruses (31/12/2009)M02-03 Submit a report on the antigenic relationships between lyssaviruses (31/03/2010)M02-04 Apply the antigenic cartography software on the Serbian panel (30/09/2011)

The antigenic cartography methods developed under SE0420 have been applied to representatives of African lyssaviruses and emerging lyssaviruses from Eurasia. A member of the team travelled to the CDC, Atlanta to study the cross neutralisation of the emerging lyssavirus Aravan (ARAV), Khujand (KHUV), Irkut (IRKV) and West Caucasian Bat Virus (WCBV). Antisera to the Eurasian viruses was raised in rabbits and used to determine the antigenic relationships between RABV and the Eurasian lyssaviruses. In addition, antisera has been raised in rabbits against the African lyssaviruses Mokola (MOKV), Lagos Bat (LBV) and Duvenhage (DUVV) Milestone 02-01. The study visit to CDC was supported by the Defra funded CIDC Programme. The antisera was used in a series of modified FAVNs to establish cross neutralisation titres of the lyssaviruses , Milestone 02-02.

We have described the antigenic relationships among a panel of 25 lyssaviruses using serological binding assay data and antigenic cartography. This approach has quantified clinically important antigenic differences between lyssaviruses; shown that those differences are equivalent for mouse, rabbit, and pooled human sera; and allowed integration of quantitative antigenic data with genetic distances. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses.

Integration of genetic and antigenic data allows identification of viruses where there are differences between genetic and antigenic relationships. For example, the antigenic positions of IRKV and ARAV, which are closer to EBLV-2, and KHUV, which is closer to RABV would not be expected from genetic relationships. Phylogenetic analysis using the glycoprotein ectodomain, suggests that KHUV is more closely related to EBLV-2 than ARAV or IRKV is to EBLV-2. Milestone 02-03

Utilising cartography software to investigate Balkan Glycoprotein sequences: Milestone M02-04The rabies virus (RABV) glycoprotein is the major antigenic component of the virus, and similarity between glycoprotein sequences is therefore a crude but simple measure of antigenic difference. Amino acid maps are a way to visualise the relationships among viruses based on this amino acid sequence diversity. Although these ‘amino acid’ maps do not incorporate evolutionary models they are useful to investigate temporal or geographic


patterns in antigenic diversity.Partial glycoprotein ectodomain sequences for twenty five representative viruses from the Balkans, and eight control viruses were aligned using Clustal X2. Amino acid sequences were derived from these sequences. The same multidimensional scaling techniques used for antigenic cartography were applied to a distance matrix generated from the number of amino acid substitutions between viral glycoprotein sequences. Amino acid sequence maps are robust and repeatable, with good self-consistency between optimisations. The glycoprotein amino acid sequence diversity among this panel of viruses from the Balkans is comparable to that among control sequences from elsewhere in Europe. There is no obvious pattern of antigenic evolution (as measured by glycoprotein identity) over time, with the largest cluster of similar viruses containing representatives from the 1970s, 1980s and 1990s. All viruses on the map isolated after 2000 are, however, in one region of the map suggesting the possible development of an antigenically distinct cluster. Comparison of glycoprotein similarity with isolation host gives a similar lack of distinct pattern. Viruses isolated from foxes are spread throughout the map, with those from ‘spill-over’ hosts interspersed. The few viruses from dogs are also dispersed on the map. Finally, comparison of amino acid similarity with the region viruses were isolated (Figure) shows that viruses from the most frequently represented region, Northern Serbia, are spread throughout the map. The isolates from Bosnia Herzgovinia are closer to each other than any other viruses, but the remaining viruses from Central/Southern Serbia, and Montenegro are also spread throughout the map. The full implication of these results for antigenic evolution are limited by the disproportionate affect of some amino acid substitutions on antigenicity. However, the lack of distinct clusters of viruses over time or by region or host, concurs with previous analyses using neutralisation data, where genetically distinct RABV are antigenically similar (Horton et al 2010). In those previous analyses, viruses from Serbia were shown to be antigenically indistinct from viruses from other regions of Europe with even higher amino acid diversity.

Figure 2. Amino acid diversity by region. Viruses (circles) are positioned relative to each other using target distances derived from glycoprotein ectodomain sequence and are coloured by region isolated (Northern Serbia=blue, Central/Southern Serbia = green, Bosnia-Herzegovina= orange, Montenegro=red). Control viruses are included (white circles)

03 To investigate quasispecies in EBLV-2 by performing in vitro and in vivo serial passages

Milestones for this objective:M03-01 Obtain genome sequence data for a bat derived EBLV-2 virus isolate (31/10/2008)M03-02 Optimise methods for detection of quasispecies sequence variants (31/03/2010)M03-03 Analyse data from in vitro passage study and perform partial genomic data comparison (30/09/2010)M03-04 Analyse data from in vivo passage study and perform partial genomic data comparison (28/02/2011)M03-05 Optimise NGS sample preparation methodologies and analyse genomic data (31/03/2012)

The genome sequence has been obtained for a bat derived EBLV-2 isolate (Milestone M03-01). This bat derived sequence was compared to the genome sequence derived from a human EBLV-2 isolate and regions of heterogeneity determined.

Quasispecies have been implicated as a possible mechanism in generating rabies virus heterogeneity. Although the heterogeneity of a RABV street-strain European fox isolate has been characterised previously, it is not known if this mechanism is used by EBLV-2. The aim of milestone M03-02 was to help optimise methods for the


detection of quasispecies so that we can further study the mechanisms in subsequent milestones.

To establish the potential for heterogeneous viral populations and host adaptation, we have analysed the consensus sequence of viruses after multiple passages for a classical rabies virus (RABV). The selected virus was isolated from a 12yr old girl who had died in Nepal after contracting rabies from a dog bite. In collaboration with Dr Ganesh Pant (Nepal), the virus was recovered from the brain of the patient post-mortem and then passaged in both mouse brain (10 passages) and in tissue culture (6 passages in BSR cells). RNA was extracted from original and passaged material and amplified by RT-PCR. Sequence data from a 405bp region of the N gene was analysed.

Multiple mouse passages appeared to have no effect on the 405 base pair region of the nucleoprotein i.e. the sequence from original brain material was identical to that after 10 passages in mice. However there was a single synonymous nucleotide transition at nucleotide position 69 (G-A) in the 6 th tissue culture passaged virus (RNA was extracted from all samples in duplicate, and both TCSN samples showed the same mutation). The nucleotide substitutions, seen in the consensus sequence from RNA extracted from the tissue culture supernatant (TCSN) but not in mouse passaged virus, contrasts that observed in a previous study which reported identical consensus sequence after 20 passages in BSR cells and one nucleotide substitution after three passages in adult mice (Kissi et al 1999). The same mutation in separately extracted RNA from the same TCSN makes PCR error extremely unlikely.

We have also determined the effect of multiple tissue culture (in vitro) passages on the glycoprotein ectodomain of a number of RABV. A rabies virus isolate (RV313) collected from a red fox in Germany showed one non-synonomous substitution at position 56 after 5 passages in tissue culture; whereas RV437 (from an Estonian raccoon dog) had two substitutions at positions 9 and 37 after 5 passages in tissue culture and RV61 (Human UK ex India) had two substitutions at positions 51 and 456 after two passages in tissue culture. This work has highlighted a high rate of non-synonomous change in the consensus sequences (relative to those seen in the work carried out by Kissi et al 1999), and in contrast to work performed with passaged influenza virus (where certain mutations tend to become fixed after passage), there is no consistent base substitution after passaging between different viruses. This data may reflect the presence of quasispecies within these isolates rather than a systematic mutational response to passage.

We have analysed the full genome sequence for RV1333 original material verses cell cultured passaged material, obtained using Sanger sequencing. In 11930bp, only 2 differences were detected: Matrix-gene T2967-C2967 which resulted in an amino acid substitution S-L and in the L-gene C9931-T (synonymous). For this reason both these regions were chosen for further quasispecies analysis. In addition the G-L region was also targeted. This region is the most variable in the genome (Marston et al 2007) and is used for phylogenetic analyses of highly similar virus isolates (Zulu et al 2009). As it is non-coding, the length of this region varies between isolates as well as the sequence diversity.

We have concentrated on a series of passaged material from an EBLV-2 isolate RV1332 which has been well characterized in both in vivo and in vitro experiments. In this series the virus is passed through both mouse brain and tissue culture, therefore putting the virus under selected pressure, the material is:

Samples highlighted in red are the samples analysed for presence of quasispecies.

Neither position 2967 nor 9931 had any changes between the passaged material. Neither did the G-L region. However, there was 1 difference observed by sequencing the PCR products, a change to the P/M transcription termination sequence from TGAAAAAAACA to TAAAAAAAACA. This was only seen in the N2A P3 sample, although when the N2A P2 chromatograms were studied, an A peak was seen in addition to the dominant G peak indicating a mixed population of viruses in that sample. Interestingly, this transcription termination sequence has been recorded to have been altered in lab attenuated strains of rabies virus (Warrilow et al 2002, Ito et al 2001), suggesting that this alteration is a feature of the adaptation of the virus to cell culture (Milestones M03-02, 03 and 04)


Original brain – MBP1 – MBP2 – MBP3

N2A P1

N2A P2

N2A P3

Although we were able to gain an understanding of quasispecies using the above methods, they were hugely time (and therefore money) consuming and ultimately the data obtained still wasn’t sufficient to fully understand the importance of quasispecies in lyssavirus adaptation. Therefore in the final year of this project we investigated the use of Next generation sequencing (NGS) technologies. The availability of a 454 and Ilumina machine at AHVLA in the Central Sequencing Facility run by experienced staff has been invaluable in this process. NGS enables investigation of quasispecies at a level that is impossible using the current cloning methodology followed by Sanger sequencing. Moreover, NGS can be applied to sequence entire viral genomes to obtain ultra deep coverage rather than having to only amplify small regions of the genome, therefore no variation is missed. NGS has the potential to provide information beyond the consensus for a viral sample by revealing nucleotide substitutions present in only a small fraction of the population. Several studies have previously used the 454 pyrosequencing platform (Roche Applied Science) to detect minority sequence variants for human viruses such as HIV-1.

Optimising NGS sample preparation methodologies and analyzing RABV samples Milestone M03-05

We have successfully developed two full genome sequencing methodologies: 1) Consensus Sequencing and 2) amplicon ‘deep’ sequencing. The consensus sequencing method is applied to obtain sequence an unknown virus (or virus for which full genome sequence is not currently available). RNA is extracted from original brain material or tissue culture supernatant without using primers. The deep sequencing method utilises overlapping PCR amplicons designed against a known virus genome sequence to ‘deep sequence’ a virus genome to investigate quasispecies.1) Consensus SequencingThis method is necessary for gaining full genome sequences from novel lyssaviruses such as IKOV or BBLV but also for viruses within a known lyssavirus species (such as EBLV-2 or RABV) because even if there are full genome sequences available for that species, the particular variant requiring full genome sequence may be significantly different to those published. This method will replace the genome walking method employed in SE0420 (the ROAME preceding this project) to sequence EBLV-1 and EBLV-2 (Marston et al 2007). Genome walking the two lyssaviruses took hundreds staff time hours and cost thousands of pounds in Sanger sequencing. In comparison, 454 consensus sequencing requires 1 day RNA prep and costs approximately £1500 to prepare and run on the machine (assuming its run alongside other samples). By searching the literature and discussing with colleagues within the AHVLA virology department we have designed a universal protocol to obtain high quality RNA from both brain tissue samples and tissue culture supernatant samples. The method from brain samples involves extracting RNA using TRIzol, then depleting gDNA and rRNA in enzymatic reactions resulting in purified RNA. The method from tissue culture supernatant involves precipitating the virus particles using PEG, then extraction of tRNA using TRIzol LS. An experiment to determine the requirement of downstream depletion of gDNA and rRNA was performed, and indicated that downstream purification was not necessary for TCSN extracted samples. This is not surprising when considering the purity of the virus in the TCSN with very low levels of cellular material. Finally, using the cell pellet obtained when clarifying the TCSN can also be used as a source of virus and is processed in the same way as a brain tissue sample. The highest amount of purified RNA was obtained using this method and full genome sequence was obtained (see table below).

We have had exceptional success using the consensus sequence methodologies with 11 full length genomes (10 Lyssaviruses) obtained by the end of this project (see table below). We have obtained full genome sequence from as little as 1.5ng/ul of purified RNA. The quality of the starting brain material is the most critical factor in the process, freshly sampled and correctly stored material provides the highest quality RNA and therefore full genome sequence with ease. Whereas brain samples stored in inadequate conditions (-20oC in African countries where electricity supply to freezers is unreliable, followed by lengthy delays during shipment to the UK without dry ice) result in failure to obtain significantly high reads to obtain good coverage. IKOV is a good example of these issues. The first sample we received from Tanzania was stored in glycerol in an ultra centrifuge which fluctuated in temperature. It had been freeze/thawed a number of times previously. We were unable to obtain NGS sequence using our methodologies. A second sample obtained from a section of freshly frozen brain which had remained in the freezer was put into RNAlater and shipped on dry ice, from which full genome sequence was easily obtained with our protocols.


Sample Sample Typeng/ul 1:10 extraction

Dnase treatment

(ng/ul)

Ribosomal depletion

(ng/ul) Full sequence?RV437 Estonian Racoon-dog Mouse Brain 540.44 410.77 165.54 Yes

RV2417 Sri-Lankan Dog Original brain 543.13 1799.21 735.1 YesSub1202 Moroccan Dog Original brain 183.39 3.27 2.15 YesSub1643 Tanzanian Dog Original brain 238.58 111.81 55.11 Yes Sub2240 Iraq dog strain (cow) Original brain 189.12 45.84 18.11 about 50%RV50 A Arizonan Bat Mouse Brain 218.62 18.76 8.13 Yes

RV2324 A Egyptian Dog (Africa 4) cell pellet 1023.29 870.72 588.86 YesRV1423 EBLV-1 TCSN 373.26 2.8 1.53 YesRV1787 EBLV-2 TCSN 460.3 37.87 13.39 about 90%

Sub2440 IKOV IKOV orig brain in RNAlater 653.74 66.55 25.95 Yes

The analysis of the raw data using de novo assembly (without reference sequence), for most of these sequences was sufficient to obtain full coverage. This is important when working with novel viruses such as IKOV, where no reference sequence is available. Establishing this technique is a significant achievement, and puts us in an advantageous position within the virology field. Certainly within the lyssavirus field there are relatively few full genome sequences published (see table below generated by Lyssavirus Sequence Database), and those that are have all been obtained using non-NGS technologies. We intend to publish this methodology in the follow-on project SE0427.

Lyssavirus speciesNumber of full genome sequences

RABV 46 street-strains, 18 vaccine/fixedLBV 4

MOKV 3DUVV 2

EBLV-1 5EBLV-2 2ABLV 2

Table showing the number of full genome sequences available on Genbank. All other species (ARAV, IRKV, KHUV, WCBV, SHIBV, BBLV) have 1 full genome published

2) Amplicon ‘Deep’ SequencingThis methodology requires the consensus sequence to be available, either by using the method above or published full genome sequences in genbank. Using the complete genome sequence, primers are designed which will produce overlapping amplicons that are subsequently sequenced on a NGS platform. The sequencing reads obtained for each amplicon will provide depth of coverage to enable analysis of minority viral populations. We have designed primers and obtained 26 overlapping PCR products to cover the entire genome of EBLV-2 (see figure below). We have obtained amplicons for the original brain material Scottish EBLV-2 Daubenton’s bat and for the original RV1333 (human Scottish EBLV-2 case). The amplified products have been sent to the AHVLA CSU facility and are currently being sequenced. The passaged material for these samples are in the process of being amplified and will be sequenced when completed. Analysis of the original and passaged data will be included in the follow on project (SE0427). Additionally, the same strategy is being employed with the Tanzanian dog genome sequence we have obtained using the consensus sequence methodology above. A PhD student co-supervised between AHVLA and Glasgow University is intending to use deep-sequencing to match contact tracking data obtained from the Serengeti in an MRC funded project.


Figure 3: Circularised schematic of EBLV-2 genome with the overlapping PCR products indicated.

04 To improve upon the VLA Lyssavirus Reference Virus Archive Milestone

Milestones for this objective:M04-01 Adapt CSF database to fulfil the needs of AHVLA rabies reference lab (31/05/2010)M04-02 Implementation of ICAST database to complement the new lyssavirus archive (31/12/2011)

Sequence Database M04-01

In collaboration with National Centre for Zoonosis Research NCZR (in particular Dr Christian SetzKorn), we have developed an online sequence database to enable easy searches of all available lyssavirus sequences with downloadable outputs (excel table or distribution map, figures 8 & 9). The rabies virus database is on-line at http://www.zoonosis.ac.uk/Rabies/Sequences/Search.

The link was sent to a number of rabies colleagues for feedback and amendments made. It took a considerable amount of time mapping the data from Nucleotide e.g. there are several ways to write 'polymerase gene' i.e. L, L gene, L-gene, Large protein, RNA Dependent RNA Polymerase plus different ways to refer to the hosts, countries etc. By mapping the variables we have reduce the chance of missed sequences and provided a universal platform for searching. The whole database will be updated regularly from Nucleotide so all updates/revisions will be caught - this is better than simply adding the most recent additions. Currently, there are just under 13,000 entries. We have incorporated the entries as they are classified in Nucleotide and ICTV. Further enhancements such as downstream applications (BLAST, alignments, phylogenetic tree) will be added to the database under SE0427.

An oral presentation was delivered at the Tripartite Meeting (AHVLA, FLI and Pasteur Inst) in December 2011 and a poster presented at Epizone Annual Meeting Virus Archive - ICAST M01-02

We originally collaborated with the HPA (ECACC) with the aim to introduce a sample management system which will track material through the various archive stages (storage, bi-products, transfer requests, QA). HPA have implemented a system known as ICAST (Inventory control and sample tracking) to track the acquisition, storage and dispatch of ECACC cell lines, viruses and reagents. ICAST is an SQL database designed in partnership with the HPA and ECACC, to enable quick data entry and tracking of millions of samples. ECACC is committed to using ICAST therefore system support will be maintained. It is 21 CFR part 11 compliant. When an item is deleted


http://www.zoonosis.ac.uk/Rabies/Sequences/Search

or requested it is date stamped enabling a full audit trail. Data entry can be by barcode, keyboard or voice recognition. Existing databases in Excel or Access can easily be imported into ICAST. Its flexibility of 5 tiers which can be labelled to suit individual storage and 10 flexible fields for sample information means that ICAST can accommodate any storage information. The information stored in the lyssavirus sequence database developed under M04/01, has been moved across to the ICAST database so that a full record of each sample is maintained. The final stage of cataloguing how much of each virus we have had been postponed until the new containment facility was commissioned, however we are in the process of achieving this currently.


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.

Freuling C, Johnson N, Marston DA, Selhorst T, Geue L, Fooks AR, Tordo N, Müller T. (2008) A random grid based molecular epidemiological study on EBLV isolates from Germany. Dev Biol (Basel). 2008;131:301-9. http://www.ncbi.nlm.nih.gov/pubmed/18634492?ordinalpos=9&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

McElhinney LM, Marston DA, Freuling CM, Cragg W, Stankov S, Lalosevic D, Lalosevic V, Müller T, Fooks AR (2011).Molecular diversity and evolutionary history of rabies virus strains circulating in the Balkans. J Gen Virol.92(Pt 9):2171-80. Epub 2011 Jun 1 http://vir.sgmjournals.org/content/92/9/2171.full.pdf+html

McElhinney LM, Marston DA, Stankov S, Tu C, Black C, Johnson N, Jiang Y, Tordo N, Müller T, Fooks AR. (2008) Molecular epidemiology of lyssaviruses in Eurasia. Dev Biol (Basel).131:125-31http://www.ncbi.nlm.nih.gov/pubmed/18634471?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Johnson N, Freuling C, Vos A, Un H, Valtchovski R, Turcitu M, Dumistrescu F, Vuta V, Velic R, Sandrac V, Aylan O, Müller T, Fooks AR. (2008) Epidemiology of rabies in Southeast Europe. Dev Biol (Basel).131:189-98http://www.ncbi.nlm.nih.gov/pubmed/18634479?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum

Geue L, Schares S, Schnick C, Kliemt J, Beckert A, Freuling C, Conraths FJ, Hoffmann B, Zanoni R, Marston D, McElhinney L, Johnson N, Fooks AR, Tordo N, Müller T. (2008) Genetic characterisation of attenuated SAD rabies virus strains used for oral vaccination of wildlife. Vaccine. 26(26):3227-35http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD4-4SC65YB-4&_user=3626285&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000061004&_version=1&_urlVersion=0&_userid=3626285&md5=d47bf2697bfc89be80becf70d392d5f9

Markotter W, Van Eeden C, Kuzmin IV, Rupprecht CE, Paweska JT, Swanepoel R, Fooks AR, Sabeta CT, Cliquet F, Nel LH.(2008) Epidemiology and pathogenicity of African bat lyssaviruses. Dev Biol (Basel). 131:317-25 http://www.ncbi.nlm.nih.gov/pubmed/18634494?ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum

Marston DA, McElhinney LM, Ali YH, Intisar KS, Ho SM, Freuling C, Müller T, Fooks AR. (2009) Phylogenetic analysis of rabies viruses from Sudan provides evidence of a viral clade with a unique molecular signature. Virus Res. 145(2):244-50. Epub 2009 Jul 21.

http://www.sciencedirect.com/science/article/pii/S0168170209002457

Fooks AR, Harkess G, Goddard T, Marston DA, McElhinney L, Brown K, Morgan D, Paul R, Thomas PJ, Smith B. (2008) Rabies virus in a dog imported to the UK from Sri Lanka. Vet Rec. 162(18):598.http://veterinaryrecord.bvapublications.com/cgi/reprint/162/18/598

Johnson N, Fooks A, McColl K. (2008) Human rabies case with long incubation, Australia [letter]. Emerg Infect Dis.14 (12) 1950-1951. http://www.cdc.gov/EID/content/14/12/1950.htmHayman DT, Johnson N, Horton DL, Hedge J, Wakeley PR, Banyard AC, Zhang S, Alhassan A, Fooks AR (2011). Evolutionary history of rabies in Ghana. PLoS Negl Trop Dis.5(4):e1001.http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001001Pant GR, Horton DL, Dahal M, Rai JN, Ide S, Leech S, Marston DA, McElhinney, LM, Fooks AR. (2011)


http://www.plosntds.org/article/info%3Adoi%2F10.1371%2Fjournal.pntd.0001001

http://www.cdc.gov/EID/content/14/12/1950.htm

http://veterinaryrecord.bvapublications.com/cgi/reprint/162/18/598


http://www.ncbi.nlm.nih.gov/pubmed/18634494?ordinalpos=8&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum



http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TD4-4SC65YB-4&_user=3626285&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000061004&_version=1&_urlVersion=0&_userid=3626285&md5=d47bf2697bfc89be80becf70d392d5f9



http://www.ncbi.nlm.nih.gov/pubmed/18634479?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum




http://vir.sgmjournals.org/content/92/9/2171.full.pdf+html




Characterization of rabies virus from a human case in Nepal. Arch Virol. 156(4):681-4. http://www.ncbi.nlm.nih.gov/pubmed?term=pant%20nepal%20rabies

Johnson N, Mansfield KL, Marston DA, Wilson C, Goddard T, Selden D, Hemson G, Edea L, van Kesteren F, Shiferaw F, Stewart AE, Sillero-Zubiri C, Fooks AR (2010). A new outbreak of rabies in rare Ethiopian wolves (Canis simensis). Arch Virol. 155(7):1175-7. http://www.ncbi.nlm.nih.gov/pubmed?term=johnson%20ethiopian%20wolves%20rabies

L.M. McElhinney, D.A. Marston, S. Leech, C. Freuling, W.H.M. van der Poel, J. Echevarria, S. Vázquez-Moron, D.L. Horton, T. Müller, A.R. Fooks. (2012). Molecular Epidemiology of Bat Lyssaviruses in Europe. Zoonoses and Public Health. in press.

D. A., Marston, D. L. Horton, C. Ngeleja, K. Hampson, L. M. McElhinney, A. C. Banyard, D. Haydon, S. Cleaveland, C. E. Rupprecht, M. Bigambo, A. R. Fooks, and T. Lembo (2012). Ikoma Lyssavirus: Highly Divergent Novel Lyssavirus in an African Civet. Emerging Infectious Diseases 18(4): 664-667http://wwwnc.cdc.gov/eid/ahead-of-print/article/18/4/11-1553_article.htm

Horton DL, McElhinney LM, Marston DA, Wood JL, Russell CA, Lewis N, Kuzmin IV, Fouchier RA, Osterhaus AD, Fooks AR, Smith DJ. (2010) Quantifying antigenic relationships among the lyssaviruses. J Virol. 84(22):11841-8. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2977894/?tool=pubmed

Marston, D.A., McElhinney, L.M., Johnson, N., Muller, T., Conzelmann, K.K., Tordo, N. and Fooks, A.R. (2007). Comparative analysis of the full genome sequence for European Bat Lyssavirus type-1 and type-2 with other lyssaviruses and evidence for a conserved transcription termination and polyadenylation motif in the G-L 3’non-translated region. J. Gen Virol. 88(4): 1302-14. http://vir.sgmjournals.org/cgi/content/full/88/4/1302

Other publications associated with SE0423

Turcitu MA, Barboi G, Vuta V, Mihai I, Boncea D, Dumitrescu F, Codreanu MD, Johnson N, Fooks AR, Müller T, Freuling CM. Molecular epidemiology of rabies virus in Romania provides evidence for a high degree of heterogeneity and virus diversity. Virus Res. 2010 Jun;150(1-2):28-33. Epub 2010 Feb 21 http://www.sciencedirect.com/science/article/pii/S0168170210000754

Zhang S, Zhao J, Liu Y, Fooks AR, Zhang F, Hu R. Characterization of a rabies virus isolate from a ferret badger (Melogale moschata) with unique molecular differences in glycoprotein antigenic site III. Virus Res. 2010 May;149(2):143-51. Epub 2010 Jan 28. http://www.sciencedirect.com/science/article/pii/S0168170210000365

Johnson N, Un H, Fooks AR, Freuling C, Müller T, Aylan O, Vos A. Rabies epidemiology and control in Turkey: past and present. Epidemiol Infect. 2010 Mar;138(3):305-12. Epub 2009 Oct 9. Review. http://www.ncbi.nlm.nih.gov/pubmed/19814851

Un H, Johnson N, Vos A, Muller T, Fooks AR, Aylan O. Genetic analysis of four human rabies cases reported in Turkey between 2002 and 2006. Clin Microbiol Infect. 2009 Dec;15(12):1185-9. Epub 2009 Jun 22. http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2009.02791.x/full

Müller T, Bätza HJ, Beckert A, Bunzenthal C, Cox JH, Freuling CM, Fooks AR, Frost J, Geue L, Hoeflechner A, Marston D, Neubert A, Neubert L, Revilla-Fernández S, Vanek E, Vos A, Wodak E, Zimmer K, Mettenleiter TC. Analysis of vaccine-virus-associated rabies cases in red foxes (Vulpes vulpes) after oral rabies vaccination campaigns in Germany and Austria. Arch Virol. 2009;154(7):1081-91. Epub 2009 Jun 12. http://www.springerlink.com/content/e3234h8757212975/

Vos A, Freuling C, Eskiizmirliler S, Un H, Aylan O, Johnson N, Gürbüz S,Müller W, Akkoca N, Müller T, Fooks AR, Askaroglu H. Rabies in foxes, Aegean region, Turkey. Emerg Infect Dis. 2009 (10):1620-2 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866395/?tool=pubmed

Harkess G, Fooks AR. Lyssaviruses: special emphasis on rabies virus and other members of the lyssavirus genus. Methods Mol Biol. 2011;665:279-307. http://www.ncbi.nlm.nih.gov/pubmed/21116807

Hoffmann B, Freuling CM, Wakeley PR, Rasmussen TB, Leech S, Fooks AR, Beer M, Müller T. Improved safety for molecular diagnosis of classical rabies viruses by use of a TaqMan real-time reverse transcription-PCR "double check" strategy. J Clin Microbiol. 2010 Nov;48(11):3970-8.


http://www.ncbi.nlm.nih.gov/pubmed/21116807

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2866395/?tool=pubmed

http://www.springerlink.com/content/e3234h8757212975/

http://onlinelibrary.wiley.com/doi/10.1111/j.1469-0691.2009.02791.x/full




http://vir.sgmjournals.org/cgi/content/full/88/4/1302


http://wwwnc.cdc.gov/eid/ahead-of-print/article/18/4/11-1553_article.htm

http://www.ncbi.nlm.nih.gov/pubmed?term=johnson%20ethiopian%20wolves%20rabies

http://www.ncbi.nlm.nih.gov/pubmed?term=pant%20nepal%20rabies


Johnson N, Vos A, Freuling C, Tordo N, Fooks AR, Müller T. Human rabies due to lyssavirus infection of bat origin. Vet Microbiol. 2010 May 19;142(3-4):151-9. http://www.sciencedirect.com/science/article/pii/S0378113510000659

Turcitu MA, Barboi G, Vuta V, Mihai I, Boncea D, Dumitrescu F, Codreanu MD, Johnson N, Fooks AR, Müller T, Freuling CM. Molecular epidemiology of rabies virus in Romania provides evidence for a high degree of heterogeneity and virus diversity. Virus Res. 2010 Jun;150(1-2):28-33. http://www.sciencedirect.com/science/article/pii/S0168170210000754

Zhang S, Zhao J, Liu Y, Fooks AR, Zhang F, Hu R. Characterization of a rabies virus isolate from a ferret badger (Melogale moschata) with unique molecular differences in glycoprotein antigenic site III. Virus Res. 2010 http://www.ncbi.nlm.nih.gov/pubmed/20109507






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