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Evidence Project Final Report

EVID4 Evidence Project Final Report (Rev. 06/11) Page 1 of 18

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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 SE0797

2. Project title

Investigation of the molecular basis for ND virulence to allow improved assessment of risk factors for infection

3. Contractororganisation(s)

Animal and Plant Health AgencyWoodham LaneNew HawAddlestoneSurrey KT15 3NB

54. Total Defra project costs £575,504(agreed fixed price)

5. Project: start date................ 1st January 2012

end date................. 31st March 2016


6. It is Defra’s intention to publish this form. Please confirm your agreement to do so.....................................................................................YES NO X(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 In these cases, such information should be detailed in a separate annex (not to be published) so that the Evidence Project Final Report can be placed in the public domain.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.

Newcastle disease, one of the most devastating diseases of poultry, is caused by Newcastle disease virus (NDV), the virulent form of avian avulavirus type 1 (AAvV-1). It is distributed worldwide and has the potential to cause large economic losses in the poultry industry. It has been reported to infect over 250 species of birds and others which have not yet been identified. This area of study provided the opportunity to address the variable threat of introduction and spread of NDV to multiple poultry species and to improve the probability that the UK remains free from this exotic disease. Biological data generated aimed to provide scientifically robust baseline data to inform risked based analyses to refine cost effective surveillance resulting in improved threat detection. As a result, this will help to ensure a sustainable UK poultry industry and that food production as well as export opportunities are not compromised.

The aim of this study was to explore factors that lead to the highly variable disease presentations observed in different species infected with NDV and ascertain if these factors contribute to the maintenance or emergence of virulence. The virulence of NDV is dependent on a number of factors with the F protein cleavage site sequence and the intracerebral pathogenicity index (ICPI) being the principle determinants of virus virulence. The V protein has also been shown to play a role in virulence through the antagonism of type 1 interferon (IFN-1) responses and may also play a role in host restriction of NDV. A panel of ten AAvV-1/NDV isolates representing the full range of genetic diversity and virulence (avirulent, lentogenic AAvV-1 to velogenic NDV) isolated from a range of susceptible hosts (poultry and wild waterfowl) were selected to assess their ability to antagonise IFN responses and determine their host tropism in vitro. Objectives 1 and 2 involved nucleotide sequencing for each isolate, to generate the amino acid sequence for the V-protein, compare phylogenetically and identify key amino acids which may relate to their ability to antagonise the interferon pathways. Within Objective 3, suitable cell lines were identified and sourced from different avian hosts species which included primary cells derived from chickens, turkeys, and ducks, and these were used to characterize the viruses in vitro, to determine virus titre in each cell line, and compare to the intracerebral pathogenicity index (ICPI) which is an indicator of virulence. These cell cultures were then stimulated with Poly(I:C) (a potent IFN-1 inducer that mimics the effects of naturally occurring dsRNA) to induce an IFN response to evaluate the viral replication characteristics and host range variability of the selected isolates. To evaluate the viral growth characteristics in the presence of IFN induced by poly(I:C), four treatments were produced: cells infected in the presence or absence of poly(I:C) and two controls of uninfected, poly(I:C) treated and untreated controls were examined. Tissue culture supernatants and cells were harvested 6-hourly over a period of 72 hours and the presence of viral RNA and IFN mRNA was measured by a semi-quantitative L gene and IFN-β real-time RT-PCR respectively. The presence of IFN-β protein was also measured by a commercially available ELISA. The results of the growth studies showed an overall indication that the


virulent virus isolates have the ability to overcome the hosts IFN response. In contrast, the two avirulent isolates were unable to overcome this antiviral/host response in any of the cell types assessed in this study. To determine whether the ability of NDV to antagonise IFN-1 correlates with mutation including amino acid substitutions, an in silico assessment was undertaken to compare the sequences of each of the ten AAvV-1/NDV isolates. Several amino acid substitutions were identified by next-generation sequencing (NGS) in key domains of the V protein of both virulent and avirulent strains corresponding with their ability to antagonise IFN responses. However, to confirm the importance of these individual or combinations of amino acid changes, a reverse genetic assessment using mutant viruses would need to be undertaken which is out of the scope of this project. These findings contribute to the improved understanding of the potential of NDV to cause disease (virulent vs avirulent), thus informing risk assessments of viruses currently circulating in Europe and contributing to the control of this notifiable disease.

This project has addressed the key policy tenants:

(i) Improvement in our ability to predict or control incursionsSeveral V protein sequences have been generated and when considered with the infectivity and replication data for these viruses, we have been able to demonstrate a means to predict the ability of particular NDV strains to infect different poultry species. Additionally, we have shown clear evidence of host restriction for the lineage 4 viruses, including the pigeon paramyxovirus (PPMV) lineage 4b where duck and turkey cells did not show susceptibility. These are important new developments and will be applied to our horizon scanning for emergent viruses.

(ii) Reduction in costs to either government or industryEvidence of host restriction and the propensity of certain viruses to block host interferon (IFN) pathways, thus displaying the potential to achieve full virulence in certain avian hosts were observed within this project. This will allow for a risk based assessment on the potential of isolates, identified in surveillance programs and following incursions, to cause notifiable disease. Rapid, risk-based and targeted responses based on scientific evidence would therefore offer the potential to mitigate the burden of notifiable disease and ultimately the cost to government and poultry industries alike.

(iii) Improvement in earlier detection of infectionAlthough the data produced in this project, will not directly improve early detection of infection, the outputs of this project will enhance the response to incursions of NDV into the UK, thus aiding in the control of notifiable disease through the reduction of spread of NDV in a risk-based manner.

(iv) Evidence base to support a future reduction in the length of time businesses remain under official restrictionsAs described above, we have shown clear evidence of host species susceptibility for certain NDV isolates and the potential of these strains to produce disease in UK poultry through manipulation of host immune responses. The algorithms developed in this study could also contribute to the understanding of this NAD and newly emergent virus strains identified in screening programs without overt risk. Policy decisions on the rigour of control measures can use these outputs as part of a science based evidence to inform risk assessments when incursions of NDV into the UK occur, thus supporting the potential to reduce the duration that businesses remain under official restrictions and trade embargoes.

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).

Objective 1: Development of molecular tools and determination of nucleotide sequence data of selected viruses

The AAvV-1 genome is approximately 15kb in length and consists of six genes which encode for six major proteins: nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), haemagglutinin-neuraminidase (HN) and a large polymerase (L). They are arranged in the order 3’-NP-P-M-F-HN-L-5’. Like other paramyxoviruses, NDV produces two additional non-structural proteins V and W that are generated by RNA editing during P gene transcription (Steward et al., 1993).

The virulence of NDV is dependent on a number of factors with the F protein cleavage site sequence (amino acid residues 113-117) being the principle determinant of virus virulence. However, it has been demonstrated that other regions of the NDV genome contribute to the virulence and pathogenicity of the virus. The V protein has been shown to play a role in virulence through the antagonism of IFN-1 responses and may also play a role in host restriction for NDV. The pathogenicity of NDV can be determined on the basis of various properties such as mean death time (MDT) in embryonated fowls eggs, intravenous pathogenicity index in six week old chickens, however based on international agreement a definitive assessment of virus virulence is based on the intracerebral pathogenicity index in day old chickens. The variation in virulence of different AAvV-1 isolates is reflected in the index ranges from 0.0 (avirulent) to 2.0 (highly virulent). The ability of this virus to cause such a variable disease presentation has been attributed to a number of factors, including the host species, age and health status. AAvV-1/NDV is a group of diverse and continiusly evolving genotypes that are divided into six broadly distict groups (lineages 1 – 6) (Aldous et al., 2003) based on their nucleotide sequences of the F gene. Addtionally, a pigeon adapted varient known as pigeon paramyxovirus (PPMV) forms a descrete group within the AAvV-1 population based on their genetic (lineage 4b) and antigenic binding properties (Aldous et al., 2004). This variant has been the cause of large ND outbreaks in the UK and continues to pose a threat to the poultry industry.

The aim of this study was to explore factors that lead to the highly variable disease presentations observed in different species infected with NDV and ascertain if these factors contribute to the maintenance or emergence of virulence. Several reports suggest that the NDV V protein, a component of the NDV genome that is involved in blocking the host immune response, has a role in virus virulence and host specificity/susceptibility. It has also been demonstrated that the V protein is effective in blocking the immune response in a host specific manner and that mutated virus where the V protein was removed showed attenuated virulence for chickens. Through these investigations, tools and data have been generated to enable identification and evaluation of viral genetic markers that may have a role in host specificity.

The aim of objective 1 was to develop molecular tools and determine the nucleotide sequence data of selected AAvV-1 isolates to identify key amino acids within the V protein. In year 1, a panel of viruses was selected from the EU/OIE/FAO International Reference Laboratory (IRL) for Newcastle disease (ND) in the Avian Virology group, APHA, Weybridge. These viruses (Table 1) were isolated from a diverse range of avian species and have displayed different pathogenicity (personal comminication with APHA NDV disease consultant Professor Ian Brown). Although the method for growing these viruses was evaluated and stocks were grown and quantified in year one of this project, we were unable to grow nine viruses in Baby Hamster Kidney fibroblast (BHK) cells. Attempts were made in the second reporting year to grow these viruses in African green monkey kidney epithelial cells (Vero), but this was also unsuccessful. To date, 29 viruses were grown successfully, and were aliquoted and stored at -70°C as the virus stock for use throughout the life of the project. These virus stocks were quantitated by titrating in Madin-Darby Bovine Kidney (MDBK) cells as a 50% tissue culture infectious dose (TCID50). RNA was extracted from virus stocks for next generation sequencing (NGS). A NGS method was developed for the sequencing of the phosphoprotein (P) gene. A total of ten viruses (highlighted in Table 1), where stocks were available, have been sucessfully sequenced and were selected for further work in the remainder of this project. The selection includes: (i) viruses that represent the range of AAvV-1 lineages, according to the Aldous et al., 2003 designation; (ii) a range of avian hosts from which the virus was isolated, and (iii) contains both virulent and avirulent isolates. These viruses were used for the initial screening of the ability of V protein to interfere with host interferon (IFN) response pathways. Where appropriate these sequences were submitted to the National Center for Biotechnology Information (NCBI) Genbank.

Table 1 – A panel of AAvV-1/NDV isolates representing the full range of genetic diversity, virulence and a range of susceptible hosts were selected and grown in MDBK cells. All sucessfully grown isolates were sequenced by NGS using Roche 454 Life Science technologies. The isolates highlighted in yellow were selected and assessed for their ability to antagonise IFN responses in objectives 3 and 4.


Assessment of pathogenicity of selected isolates by intracerebral pathogenicity Index test

The pathogenicity of each virus from the panel of ten AAvV-1 isolates, covering the different linages and host ranges, was assessed by the intracerebral pathogenicity index (ICPI) test as outlined in the OIE terrestrial manual (OIE, 2015). All animal work was carried out in accordance with the Virology department’s research home office license 70-8332 and approved by the animal welfare ethics committee board (AWERB). Briefly, each virus was diluted 1:10 in isotonic saline and injected intracerebrally into ten, one day-old chicks. Birds were observed three times a day over period of 8 days. At each observation the birds were scored based on the clinical signs observed (0=healthy, 1=sick, 2=dead). The index is calculated as the mean score per bird, per observation ranging from 0.0 to 2.0. An ICPI score of 0.7 or greater confirms the virulence for NDV as per the definition of Newcastle disease given in OIE Terrestrial manual. Strains of NDV are usually grouped into three pathotypes on the basis of the clinical signs seen in infected chickens. Lentogenic isolates of low virulence cause mild respiratory or enteric infections, viruses of intermediate virulence that cause respiratory disease are termed mesogenic while viruses that are highly pathogenic are known as velogenic. The ICPI score for each of the selected isolates in this study are summarised in table 2 and represent the full range of pathogenicity found in poultry from avirulent APMV1/turkey/UK/2262/2008 and APMV1/teal/Finland/Li13111-2008/2013 (2262-08 and 1148-13) to velogenic APMV1/chicken/United States of America/211472/2003 (1060-03).

Objective 2: Analysis of molecular data generated and collated in objective 1

The P gene (which encodes for the V protein through RNA editing) of each of the viruses selected under objective 1 were sequenced by NGS. The NGS approach allows for the generation of the nucleotide sequence of highly divergent viruses without optimising the conditions for each virus independently. Initially, the viral sequences generated by NGS were produced using Roche 454 Life Science technologies. However, through the continued efforts of the NGS Working Group at the APHA, encompassing members of this project team, the APHA Avian Virology workgroup, Virology Department and the Central Sequencing Unit, new technologies such as sequencing by synthesis (SBS) technology by Illumina was sought and trialled. This was particularly timely as Roche are due to remove their 454 NGS technology from the market. Illumina NGS was shown to produce greater depth (the number of sequence reads obtained across a single nucleotide) of sequence data, but was still unable to produce 100% coverage of the genome. Analysis of the data produced by Illumina has proven to be far more difficult than 454 data, requiring specialist input. Several bioinformatics analysis software programs have been trialled to analyse the P gene data produced by 454 and subsequent Illumina NGS platforms. These have included GS De Novo Assemble and GS Reference Mapper (Roche), Biolinux, galaxyproject.org and SeqMan Ngen (DNAStar). These programs require significant computer hardware powerful enough to analyse the vast amount of data generated by the NGS technology. Adequate computer hardware has been sought in coordination with Dr Helen Everett within the Virology department to address this issue with some success. However, the process of data mining and analysis remains slow and very resource intensive. An in-house funded seedcorn project (EXRD0076) was used to provide a pipeline for NGS analysis of sequences produced in collaboration with the APHA central sequencing unit (CSU) has been inititated. Added value has also been obtained from the EU Horizon 2020


compare project (WP2) which has involved the refining of sequencing methodologies. This has provided added value to SE0797 and allowed data from the project to feed into this pipeline thus providing the specialist analyses required. This has improved the turnaround time and quality of the analyses produced. For the ten viruses, the V gene sequence has been obtained and aligned (Figure 1), along with a distance matrix map (Figure 2) to evaluate the relationship of each of the isolates. Phylogentic analysis illustrates that the virulent and avirulent isolates separate at a common ancester, with lineage 6 isolate showing to be from a highly divergent population. Where appropriate, these sequences have been submitted to the NCBI Genbank (output).

Figure 1 - Phylogenetic analysis of the V gene for each of the ten AAvV-1/ND isolates using Neighbour - Joining method conducted in MEGA 6 illustrating the relationship between each of the lineages selected.

Figure 2 – Distance matrix providing a two dimensional array containing the distances of each of the ten AAvV-1/ND isolates expressed as a percentage.

Objective 3: Development of in vitro tools to assess selected viruses with variable V protein mRNA, with respects to host susceptability and growth characteristics

The aim of objective 3 was to evaluate the specific biological properties of the selected viruses identified in Objective 2 using in vitro assays that were developed in this objective (3). These assays have enabled the investigation of:

Virus growth characteristics in cell cultures from selected host species.

The effects of endogenous IFN from selected hosts on virus growth characteristics.Suitable cell lines were identified and sourced from different avian hosts which included primary cells derived from chickens, turkeys, and ducks. These cell cultures were used to evaluate viral replication characteristics of the selected isolates and to identify pairs of viruses with differing phenotypes to be assessed in Objective


4.

Characterise replication properties of the selected viruses in cell culture systems of different species

The infectivity of the ten selected viruses, was examined in chicken embryo fibroblast (CEF), turkey embryo fibroblast (TEF) and duck embryo fibroblast (DEF) cells. Briefly, cells were inoculated with a 10-fold dilution of selected AAvV-1 isolates before incubation at 37°C for 3-7 days. Each well was tested for haemaglutination activity (HA) and virus titres (TCID50) were determined. These titers (table 2) were used to determine host susceptibility and calculate the multiplicity of infection (MOI) of each isolate in each cell types to be used in the subsequent experiments to determine the effects of IFN on virus replication (described below). Where a TCID50 titer was not determined, no further downstream analysis was carried out which is required for the MOI calculation.

The data illustrates that infectivity was clearly host species restricted for the lineage 4 isolates, including the pigeon paramyxovirus (PPMV) lineage 4b. The duck and turkey cells did not show any susceptibility to PPMV isolate, APMV1/pigeon/Estonia/2-06-5252-4S/2006 and the additional lineage 4 waterfowl isolate APMV1/waterfowl/Cyprus/1530/2005 was unable to infect turkey cells. All other isolates were able to infect the duck cells (9/10) with turkey cells showing less susceptibility (7/10). All isolates were able to infect chicken (CEF) cells and produced some of the highest titres suggesting these cells are highly susceptible to AAvV-1. This data provides clear evidence of the potential infectivity of these viruses for different avian species that can be used in risk assessments once incursions of NDV or PPMV into the UK occur allowing for a rapid, risk-based and focused response to control notifiable disease.

Table 2 - Infectivity as determined by TCID50 for the ten AAvV-1 isolates

Abbreviations: CEF = chicken embryo fibroblasts; DEF = Duck embryo fibroblasts; TEF = Turkey embryo fibroblasts; ICPI = Intracerebral pathogenicity Index; TCID50 = 50% Tissue culture infective dose.

The effect of endogenous interferon on the growth properties of AAvV-1/ND viruses

Evaluation and optimization of in vitro tools

Double-stranded RNA (dsRNA), is considered a pathogen associated molecular pattern (PAMP), since some viruses exist in the form of dsRNA, but also because dsRNA intermediates are produced during viral replication. Since dsRNA is not part of normal host cellular life cycle, the presence of dsRNA activates the host immune system, particularly the interferon (IFN) response. The interferon system is one of the first line of the host response against viral infections through the inhibition of viral replication to help control the spread of the virus (Fontana et al., 2008 and Goodbourn et al., 2009). Polyinosinic: polycytidylic acid [Poly(I:C)] is a synthetic dsRNA that mimics the effect of naturally occurring dsRNA and is considered the most potent IFN-1 inducer (IFNα/β) (Li et al., 2012, Huang et al., 2003) and was assessed in this study as a suitable IFN-1 inducer. A literature review was undertaken to identify a suitable real-time RT-PCR for the detection of IFN type 1 in chicken, turkey and duck species. An in-house IFN-α real-time RT-PCR that has been developed by our collaborators at Nottingham University was evaluated for our own purposes. However, a literature review confirmed that IFN-β is the dominant cytokine produced and would be the most suitable downstream test. A commercial real-time RT-PCR - chicken IFN-β Taqman® Gene expression assay (Applied Biosystems) - for the detection of IFN-β mRNA transcripts was also trialled.


A review of the scientific literature showed a clear absence of any description of transfection reagents required to deliver poly(I:C) intracellularly. Contrary to the published literature, our assessments showed that cells that had not been transfected using a transfection reagent (e.g. FuGene) did not display an increase in type 1 IFN production. The concentration of FuGene transfection reagent was optimised for the avian species fibroblasts used.Briefly, cell monolayers were transfected with FuGene® and treated with 1µg/ml of Poly(I:C) or left untreated. Complexes were formed by mixing the Poly(I:C) with transfection agent FuGene® at a 3:1 ratio and incubated for 20 minutes at room temperature before adding to the cells. Pre-treated and non-treated cells were incubated for 24 hours. After an incubation period of 24 hours a 4 ct value difference was detected by the chicken IFN-β Taqman® Gene expression assay (Applied Biosystems).

As IFN-β mRNA is only detectable within the cells, an alternative extraction process was required. Qiagen offer an RNeasy extraction kit which provides a lysis stage to enable the detection of IFN. Due to the sample numbers expected from these virus replication studies, a robot kit and software was sourced to aid throughput. However, as this protocol had not been used routinely within the workgroup, no inactivation validation was available to remove the samples from SAPO4/ACDP3 facilities safely. Validation of the inactivation of AAvV-1 by Qiagen RLT buffer was undertaken to allow the removal of these samples from the high containment facilities to laboratories of lower containment for nucleic acid extraction. Specifically this data evaluated virus inactivation during RNA extraction using Qiagen RNeasy extraction kit with RLT buffer (lysis buffer) and 70% ethanol. This study used a representative AAvV-1 isolate - APMV-1/chicken/Scotland/1453/96 - at a titre of 9.2 log10 EID50/ml. As RLT buffer is toxic to eggs an initial experiment was set up to determine the concentration at which RLT buffer/ethanol no longer causes toxicity in eggs. Eggs were candled daily and the allantoic fluid from any dead eggs was harvested and tested for HA activity. The results demonstrated that all eggs survived at a dilution of 1:8 and was subsequently used as the limit of sensitivity for the inactivation validation process. Briefly, the AAvV-1 isolate was diluted to 1:1000, 1:2000, 1:4000 and 1:10,000 in minimum essential media without sera (MEM-WOS) in 12 well plates containing CEF monolayers and incubated at 37°C. After 48 hours the supernatant was removed and RLT buffer added to each well. The RLT buffer containing cells (or cellular debris) was subsequently removed and transferred to a microcentrifuge containing equal volumes of 70% ethanol. The mixture was then inoculated into embryonated fowls eggs and incubated at 37°C for 6 days. Eggs were candled daily and after 6 days any survivors were chilled and tested for HA activity. The results provide sufficient evidence that RLT/ethanol inactivated the virus allowing the safe removal from containment facilities. The validation document has been prepared and approved by the team leaders before submitting to the in-house validation team for approval.

Additionally, the suitability of an in-house IFN-α ELISA that had been developed as part of an MSc project at the APHA was evaluated for use in this objective. A comprehensive review of the ELISA data identified that it was not suitable for our requirements and further research was undertaken. A chicken and duck IFN-β ELISAs were identified as suitable candidates and were sourced from a commercial company in the USA (BioSource). These kits allow the quantification of IFN-β from tissue culture supernatants. Although a turkey IFN-β assay is not commercially available, published data demonstrates that there is cross-reactivity between chicken and turkey type 1 IFN (Suresh et al., 1995).

Evaluation of viral and IFN production in cell cultures

Ten AAvV-1/NDV isolates representing the full range of genetic diversity and virulence (avirulent, lentogenic AAvV-1 to velogenic NDV) and isolated from a range of susceptible hosts (poultry and wild waterfowl) were selected to assess their ability to antagonise IFN responses and determine their host tropism in vitro. Chicken, turkey and duck embryo fibroblasts were stimulated by poly(I:C) to induce an IFN response and infected 24 hours later with these AAvV-1/NDV isolates at a 0.01 MOI. To evaluate the viral growth in the presence of IFN induced by poly(I:C), four treatments were produced (Table 3). Cells were infected in triplicate in the presence or absence of poly(I:C). Two controls of uninfected, poly(I:C) treated and untreated controls were produced.

Table 3 – Four treatments evaluated to assess the viral replication and IFN production in chicken, turkey and duck embryo fibroblast cells.


For the lentogenic strains 2262-08 – APMV1/turkey/UK/2262/2008 (lineage 1) and 1148-13 – APMV1/teal/Finland/li13111-2008/2013 (lineage 6), these viruses can only replicate in the presence of trypsin-like enzymes in the respiratory and intestinal tract. Therefore, to ensure replication in this cell culture system, the growth media included 1mg/ml of trypsin to facilitate the entry of the viruses into the cells. Tissue culture supernatants and cells were harvested 6-hourly over a period of 72 hours. Viral RNA was extracted from the supernatant using the Qiagen QiAMP extraction kit according to the manufactures protocol. A L gene minor groove binding (MGB) real-time RT-PCR (referred to as the MGB assay), designed as part of Defra project SE0794, was employed for the detection of AAvV-1 in supernatant samples. The presence of IFN-β was measured by a commercially available ELISA kit (BioSource). Additionally, the chicken IFN-β Taqman® Gene expression assay was used to measure the expression of mRNA present in the cells. To control for experimental variation and confirm extraction procedures, a glyceraldehyde 3-phosphate dehydrogenase (GAPDH) which is one of the most commonly used housekeeping genes for the comparison of gene expression data was used (GAPDH Taqman® Gene expression assay - Applied Biosystems) in conjunction with the IFN-β PCR.

The results in this growth study (Figure 3, 4 & 5) suggest that for virulent viruses, as viral replication increases, INFN-β production decreases. AAvV-1 RNA was detected by MGB RT-PCR after pre-treatment with poly(I:C) a delay of ~24 hours was observed when compared to the non-treated controls. All isolates which are virulent in chickens showed an increase in viral RNA after ~36 hours (Figure 3). This suggests that these viruses have the ability to overcome the IFN response in these cells. This is potentially mediated by the viral V protein, which has previously been shown to act as an interferon antagonist (Alamares et al., 2010; Irie et al., 2012; Qiu et al., 2016). However, growth of the virulent isolates in TEF (Figure 4) was relatively slow to respond except for APMV1/turkey/Finland/805/2004 (805-04) and APMV1/chicken/Romania/858/2007 (2515-07) which showed an increase in replication after ~12 hours. Observations for APMV1/partridge/UK/7575/2006 (7575-06) in DEF show no recovery or ability to replicate in the cells under Poly(I:C) treatment compared to the other virulent isolates. This may be due to the batch of virus used or a genuine result in this cell line. To confirm if this was a true result this isolate should be retested. In contrast, the avirulent isolates APMV1/turkey/UK/2262/2008 and APMV1/teal/Finland/Li13111-2008/2013 (2262-08 and 1148-13) do not show recovery in any of the three cell types assessed in this project. Interestingly, infection of CEF with the PPMV isolate, PPMV1/pigeon/Estonia/12-06-5252-45 lineage 4b, shows an increase in viral RNA not observed for this isolate previously. Historically, infection with PPMV does not cause significant disease in chickens. This suggests that the PPMV V protein of PPMV1/pigeon/Estonia/12-06-5252-45 lineage 4b was unable to fully unblock the IFN response in chickens and thus remain of lower virulence. Additionally, these isolates were originally grown in embryonated fowls eggs which may have adapted them to grow more efficiently in a galliform cell culture system.

Evaluation of the IFN-β production for all isolates in CEF by ELISA shows an overall increase of production over the 72hr growth study even if the virus was able to recover successfully. However, when compared to the infection of DEF cells (figure 5), IFN-β production for all virulent isolates decreases as viral replication increases demonstrating that the all the virulent isolates were able to antagonise the IFN-β pathways. In contrast, TEF showed a continuous production of IFN-β with only 805-04 showing evidence of interfering with the IFN pathways (Figure 4). To understand if the V protein acts on the IFN mRNA pathway we also assessed each of the time points using the IFN-β real-time PCR. The data showed a constant detection of IFN-β mRNA throughout each of the time points tested for all CEF samples demonstrating that there is no change in IFN-β mRNA production. However, when the TEF and DEF infected cells were evaluated, no IFN-β mRNA was detected. This may be in part due to the PCR not being specifically designed to target turkey or duck IFN even though cross reactivity data has been published.

When the morphology of the cell monolayers were examined, the cells in the poly(I:C) treated but uninfected treatment had died, since poly(I:C) is a potent apoptosis inducer. However, the poly(I:C) treated and infected cells remained confluent. This suggest the presence of virus is blocking the poly(I:C)-induced apoptosis pathways allowing for the continued proliferation of virus. To confirm if this process is correct a terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) staining technique would need to be undertaken for the detection and quantification of apoptosis in cell monolayers.

Figure 3 – Comparison of viral growth and interferon-β production in non-treated and Poly(I:C) pre-treated chicken embroynated fibroblast cells at 37°C. CEF’s were infected with each indicted virulent or avirulent virus at an MOI of 0.01 as determined by measuring the TCID50. Triplicate supernatants were harvested every 6 hours for 72 hours and the relationship between viral RNA (blue line) and IFN-β production (red line) were plotted. The results suggest that for virulent viruses, as viral replication increases INFN-β production decreases, demonstrating virulent isolates have the ability to antagonise the IFN pathways. In comparison, avirulent viruses do not efficiently replicate as they cannot overcome the IFN


response.


Figure 4 – Comparison of viral growth and interferon-β production in non-treated and Poly(I:C) pre-treated turkey embroynated fibroblast cells at 37°C. TEF’s were infected with each indicted virulent or avirulent virus at an MOI of 0.01 as determined by measuring the TCID50. Triplicate supernatants were harvested every 6 hours for 72 hours and the relationship between viral RNA (blue line) and IFN-β production (red line) were plotted. The results suggest that for virulent viruses, as viral replication increases INFN-β production decreases, demonstrating virulent isolates have the ability to antagonise the IFN pathways. In comparison, avirulent viruses do not efficiently replicate as they cannot overcome the IFN response.


Figure 5 – Comparison of viral growth and interferon-β production in non-treated and Poly(I:C) pre-treated duck embroynated fibroblast cells at 37°C. DEF’s were infected with each indicted virulent or avirulent virus at an MOI of 0.01 as determined by measuring the TCID50. Triplicate supernatants were harvested every 6 hours for 72 hours and the relationship between viral RNA (blue line) and IFN-β production (red line) were plotted. The results suggest that for virulent viruses, as viral replication increases INFN-β production decreases, demonstrating virulent isolates have the ability to antagonise the IFN pathways. In comparison, avirulent viruses do not efficiently replicate as they cannot overcome the IFN response.


Determination of amino acid substitutions in V protein and their ability to antagonise IFN pathways

Viral IFN antagonists have shown to be important virulence factors in several paramyxoviruses (Alamares et al., 2010, Huang et al., 2003, Park et al., 2003). To determine whether the ability of NDV to antagonise IFN-1 as observed in the growth studies correlates with amino acid substitutions, an in silico assessment was undertaken to compare the sequences of each of the ten AAvV-1/NDV isolates. During this assessment we idenified differences in the sequence of isolate 1148-13 - lineage 6 which created difficulties in aligning the sequence in Megalign (DNASTAR) with the other isolates which was in part due to being from a highly divergent population. Therefore, this sequence was excluded from this in silco assessment and 1294-07 - mallard/Germany/SU68R/2007 was included as an additional avirulent isolate. In this study, several amino acids were identified in the V protein as potential candidates (figure 6), that may contribute to IFN antagonistic activity. The amino acid substiutions determined were residues 161, 162, 188 and 201 within the C-terminal of the V protein that may contribute to the isolates ability to cause disease. Although it is widely recognised that these residues are located in the C-terminus region, several antagonistic residues (11,12, 68 and 94) were identified within the N-terminus supporting the work published by Alameres et al., 2010. Analysis of the V protein sequences in Megalign across the ten AAvV-1/ND isolates in accordance with the ICPI and growth curve studies only showed one potential candidate (residue 149) that may be specific for all three pathotypes (Figure 6). To confirm the importance of indiviaual or combinations of amino acid changes, a reverse genetic assessment using mutant viruses would need to be carried out. In addition,


there are no amino acids in the V protein that are specific for the different pathotypes (lentogenic, mesogenic or veloginic) in each of the selection of hosts assessed (chicken, turkey or duck). However, a larger panel of sequences would be required to provide a more comprehensive assessment which will form part of the ND futures program (SE2208).

Figure 6 - Representation of the amino acid substitutions in the V protein of the ten AAvV-1/NDV isolates covering the full genetic diversity and pathogenicity.

Abbreviations: A=Alanine, D=Aspartic acid, E=Glutamic acid, G=Glycine, I=Isoleucine, L=Leucine, P=Proline, Q=Glutamine, R=Arginine, S=Serine.

Figure 7 – Sequence alignment of the ten AAvV-1/ND isolates selected for assessment, highlighting residue 149 as a potential candidate that may be specific for all three pathotypes (lentogenic, mesogenic or velogenic). The findings suggest that amino acid alanine (A) are associated with avirulent strains, Serine (S) Lentogenic strains, proline with Mesogenic strains and glutamine (Q) with velogenic isolates.

Objective 4 - Biological assessment of V protein variant viruses

Throughout this project we have investigated the effects of V protein and the role it plays in antagonism of type 1 interferon pathways and host restriction. In this objective, it was proposed that a biological tool would be developed targeting the viral properties identified and their ability to antagnoise the IFN pathway to help make informed decisions and estimate the risk pathways. However, the findings observed in objective 3, provide a clear indication from the lack of IFN-β mRNA detected by the real-time PCR that the V protein acts on the IFN-β protein and not the mRNA as initally thought. This means that any biological assays developed in this objective such as co-transfecting an expression plasmid with the test virus to determine the transcrition factor that the V protein acts on would be obsolete. Additionally, the algorithim developed in objective 3 for this V protein assessment is time consuming and expensive to perform with an estimated turn around time of approximately 18 days (Figure 8). Although this process could still be used alongside the current ND alogrithim (highlighted in blue in figure 8) and provide useful information to help inform risk


assessments the turnaround time of this protocol would not make it a suitable as a front line testing method highlighting that it would be more useful as a reasearch tool.

Figure 8 – Representation of alogorithim currently employed by the APHA for suspected AAvV-1 cases. Hightlighted in blue provides the testing procedures required for complete V protein variant assessment and additional time required.

Summary and future work

In this report we have described the investigation of specific viral factors that may influence the variable disease presentation observed in different poultry host species infected with NDV. The main outcome has included that the avian host species assessed in this study have variable susceptibility to each of the selected isolates which cover the full genetic diversity of AAvV-1 with chicken cells being the most susceptible. The V protein has been shown to play a role in its ability to cause disease through the antagonism of type 1 interferon responses. We have identified several amino acid substitutions in the V protein that correspond with their ability to antagonise IFN. These mutations provide an improved understanding of the potential of NDV to cause disease, thus informing risk assessments and contributing to the control of this notifiable disease. However, further work is needed to refine key mutations and actions of V protein to confirm its role. This work also suggested that a reduction in disease outbreaks resulting from PPMV infection may also be related to V protein antagonism of IFN. Further work is therefore needed to determine the mechanism for reduced disease presentation in poultry infected by PPMV which will be undertaken in ND futures program (SE2208). The effect of individual mutations within the V protein will be assessed using mutant viruses generated using reverse genetics technologies. The ability of these mutant V proteins to bind directly to IFN or other proteins within the IFN pathway and cause disease will be assessed by co-immunoprecipitation assays thereby defining the direct action of V protein as well as in vitro (infectivity and growth assessments in chicken, turkey and duck embryo fibroblasts and confocal microscopy) and in vivo methods. This will identify key mutations in this protein affecting the ability of NDV and PPMV to cause disease.

In summary, this study has provided a better understand of the variable threat posed by different poultry


species and different virus strains for the introduction and spread of NDV. The data generated will provide information that can support risk based analysis to help refine surveillance approaches. This in turn will contribute to improved threat detection; thereby helping the UK to remain free from this exotic trans boundary viral disease and contributing to ensuring a sustainable UK poultry industry and other economic opportunities are not compromised.

References

Alamares, J. G., Elankumaran, S., Samal, S. K. & Iorio, R. M. (2010) The interferon antagonistic activities of the V proteins from two strains of Newcastle disease virus correlate with their known virulence properties. Virus Res 147, 153-157.

Aldous, E. W., Mynn, J. K., Banks, J. & Alexander, D. J. (2003) A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene. Avian Pathology 32, 239-256.

Aldous, E. W., Fuller, C. M., Mynn, J. K., & Alexander, D. J. (2004) A molecular epidemiological investigation of isolates of the variant avian paramyxovirus type 1 virus (PPMV-1) responsible for the 1978 to present panzootic in pigeons. Avian pathol 2, 258-269.

Fontana, J.M, Bankamp, B., Rota, P.A. (2008) Inhibition of interferon induction and signalling by paramyxoviruses. Immunol Rev 225, 46-47.

Goodbourn, S., Randall, R.E. (2009) The regulation of type 1 interferon production by paramyxoviruses. J Interf Cytok Res 29, 539-547.

Huang, Z., Krishnamurthy, S., Panda, A. and Samal, S.K. (2003) Newcastle disease virus V protein is associated with viral pathogenesis and functions as an alpha interferon antagonist. Journal of Virology 77, 8676-8685.

Irie, T., Kiyotani, K., Igarashi, T., Yoshida, A., Sakaguchi, T. (2012) Inhibition of interferon regulatory factor 3 activation by paramyxovirus V protein. J Virol 86, 7136-7145.

Li, Y., Siripanyaphinyo, U., Tumkosit, U., Noranate, N., A-nuegoonpipat, A., Pan, Y., Kameoka, M., Kurosu, T., Ikuta, K., Takeda, N. and Anantapreecha, S. (2012) Poly(I:C), an agonist of toll-like receptor-3, inhibits replication of the Chikungunya virus in BEAS-2B cells. Virology Journal (9)114.

Office International des Epizootics OIE (2015) Manual of diagnostic tests and vaccines for terrestrial animals. Chapter 2.3.14.

Park, M. S., Garcia-Sastre, A., Cros, J. F., Basler, C. F. & Palese, P. (2003) Newcastle disease virus V protein is a determinant of host range restriction. J Virol 77, 9522-9532.

Qiu, X., Fu, Q., meng, C., Yu, S., Zhan, Y., Dong, L., Song, C., Sun, Y., Tan, L., Hu, S., Wang, X., Liu, X., Peng, D., Liu, X., Ding, C. (2016) Newcastle disease virus V protein targets phosphorylated STAT1 to block IFN-1 signalling. PLoS One 11, e0148560.

Steward, M., Vipond, B., Millar, N.S., and Emmerson, P.T. (1993) RNA editing in Newcastle disease virus. Journal of General Virology 74, 2539-2547.

Suresh, M., Karaca, K., Foster, D. & Sharma, J.M. (1995) Molecular and functional characterization of turkey interferon. Journal of Virology 69, 8159-8163.

Zhang, J., Kaiser, M.G., Deiat, M.S., Gallardo, R.A., Bunn, D.A., Kelly, T.R., Dekkers, J.C.M., Zhou, H., Lamont, S.J. (2018) Transcriptome analysis in spleen reveals differential regulation of response to Newcastle disease virus in two chicken lines. Sci Rep 8, 1278.


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.

Conference Proceedings Abstract & Presentation

Brandon Z. Löndt, Jo Mayers, Chad Fuller, James Seekings, Sharon M. Brookes, & Ian H. Brown. The role of the V protein of Newcastle disease virus (NDV) in virulence and host restriction. Virology Africa 2015, Cape Town, South Africa.

Jo Mayers, Anita Puranik, David Sutton, Caroline Warren, Chad Fuller, James Seekings, Brandon Z. Löndt, Sharon M. Brookes & Ian H. Brown. The role of the V protein of Newcastle disease virus (NDV) in virulence and host restriction. Veterinary Research Club meeting held on 22nd April 2016, University of Surrey.

Peer-review publication in preparation

Jo Mayers, Anita Puranik, David Sutton, Caroline Warren, Chad Fuller, James Seekings, Brandon Z. Löndt, Sharon M. Brookes & Ian H. Brown. The role of the V protein of Newcastle disease virus (NDV) in virulence and host restriction.


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