A strategic approach to identifying and combating porcine reproductive and respiratory syndrome virus outbreaks and other porcine viral diseases

Lead Research Organisation: University of Edinburgh
Department Name: The Roslin Institute

Abstract

This project aims to develop a strategic approach to identify and combat porcine reproductive and respiratory syndrome virus (PRRSV) outbreaks and other porcine viral diseases in the Philippines. The Philippines are the world's 8th largest producer of pork producing over 1,700 mega tons of pork meat per year. However, the pork production still relies largely on backyard farms, which, in 2016, were reported to house 64% of the roughly 12.5 million animals in the country. This divergence in farming practises between backyard and commercial farms requires a robust strategic approach to prevent pathogen spread across farms and systems. The backyard farms often use crossbred commercial western/indigenous breed pigs, with few biosafety measures in place, rarely rely on veterinary consultation, lack diagnostics, and allow fairly liberal and unsupervised access to antibiotics. In contrast, the commercial farms rely on commercial highly bred western pig breeds, mostly Landrace/Large White/Duroc crosses brought in from leading breeding companies, such as PIC and Topigs Norsvim, amongst others. The commercial farms, despite little usage of diagnostics to support clinical diagnoses, are employing extensive vaccination schemes; however, currently this isn't always effective in preventing disease. Improved diagnostics, monitoring, surveillance and reviewing of current biosafety and vaccination strategies are essential to employ a targeted approach to disease control.

Therefore, this project aims to assess the current health situation in commercial and backyard farms, to identify current vaccination strategies, and to identify circulating strains and subtypes of PRRSV and PEDV. We will use traditional Sanger and next generation sequencing of field samples to conduct phylodynamic and phylogeographic analysis, which will help to inform the spread and source of PRRSV and PEDV outbreaks, and which will further aid disease prevention strategies. The outcomes will give us an overview of the current health and biosafety situation in the Filipino pig industry and will allow us to develop specific, easy-to-use diagnostic tools that will enable farmers to specifically identify diseases present in their stock and advise targeted vaccination and biosafety measures. We aim to develop differential diagnostic tools based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) or reverse transcription recombinase polymerase amplification (RT-RPA) for systemic viral diseases including PRRSV, CSFV, PCV2, and IAV-S, and for enteric viral diseases including PEDV, TGEV, and rotaviruses. We will employ easy-to-use flow strip detection methods to develop multiplex tests that allow differentiation between viruses and/or subtypes. From our results, we aim to generate targeted advice for both commercial and backyard farms in order to prevent and combat viral diseases in pigs.

Planned Impact

Who will benefit from this research?

The potential non-academic beneficiaries of this research include pig diagnostic labs, pig health companies, pig breeding companies, pig producers and ultimately the entire chain of users of pig products, including meat packers, processors, retailers and consumers. There are also potential benefits to the pig vaccine sector.

How will they benefit from this research?

Porcine viral disease is of major impact to pigs of all ages. Clinical manifest disease often leads to high mortality in suckling and weaned piglets and slower weight gain in animals of all ages. Therefore, this has an immediate effect on feed efficiency, productivity and the livelihood of farmers. Accurate and fast diagnostic will allow to take the correct biosafety and biosecurity measures to prevent disease spread, advise the use of appropriate vaccination where available, and in consequence improve the health of one farm and prevent spread to other farms. This benefits the pig producers, as well as the pig product chain immediately. Pig diagnostic labs and pig health companies can be involved in the commercialisation and distribution of such diagnostic tools and the pig breeding companies are benefiting from wealthier farmers that can afford high breed animals, but also their own elite and producer farms will be benefiting from these tools.
 
Description This award was severely impacted by COVID. The funding of our Filipino partners was suspended for a year to deal with the impact of COVID in the country, which meant funding had to be allocated elsewhere. The funding has restarted in June 2021 and will now last until June 2022.
As a consequence, this grant now not only been affected by the outbreak and spread of ASFV in the Philippines but also the restrictions on people's movements around the country due to COVID, which has severely affected sample collection. We have been promised archived samples of PRRSV outbreaks by BAI to aid with phylogeny and phylogeography of PRRSV and infectious disease spread in the country. However, we will only be able to obtain these samples once our Filipino partners' funding restarts and travel restrictions have been removed. We managed to allocate alternative funds to return to the Philippines in May 2022, after this grant to finalise some aspects of this grant.

We have successfully developed robust protocols to sequence and assemble full-length genomes of porcine reproductive and respiratory syndrome virus (PRRSV) using Nanopore MinION sequencing from serum samples and tissue culture amplified virus by cDNA, PCR-product, and RNA sequencing. As the full-genome sequencing was not as sensitive as it needed to be for serum samples, we have since developed an overlapping fragment amplification that was universal for all PRRSV-1 and PRRSV-2 samples tested, respectively. This has allowed us to obtain whole genome sequences of PRRSV from serum samples. Delays due to COVID on animal challenge trials (unrelated to this grant) meant we were delayed in validating some of these protocols but we are now in the process of publishing these findings. We are preparing two publications; one on tiled sequencing and assembly of PRRSV and one on whole genome sequencing and quasispecies identification.

Samples from regions IV, III, X, and V have been collected and so far 40 samples were found to be PRRSV-positive. Due to the outbreak of ASFV in autumn 2019 sampling has been disrupted. Therefore, we opted for using samples linked to the ASFV depopulation, which will give us further PRRSV-positive samples from ASFV-negative farm pools.

Since we wanted to build capability and capacity in the Philippines when visiting last time in November 2019, we took along DNA sequencing kits as well as the sequencing reagents for PRRSV. This has allowed us to obtain whole-genome sequences from ASFV samples in the Philippines as well, which has supported their efforts in developing reagents to tackle the ASFV outbreak and spread in the country. We have developed optimised whole-genome sequencing protocols, which we have published as a preprint and obtained further funding for protocol optimisation and international collaborations on this from Oxford Nanopore Technologies plc. We are finalising phologenetic and -geographic analyses including samples we obtained from other partners and will be publishing within the next couple of months.

Originally developed for PRRSV sequencing assembly, we have also released the bioinformatics pipeline Lilo. Lilo is a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. This is particularly important for viruses with hypervariable regions or high mutagenesis rates, such as PRRSV, and viruses or other pathogens with INDELs.

Reagents and protocols for multiplex porcine virus diagnostics have been developed and are currently being assessed and protocols optimised. Whilst multiplexing works in the lab we are currently putting this on hold due to the difficulties of obtaining RPA reagents in the Philippines, which makes the application of the diagnostic test difficult. Therefore, we have focused on the development of RT-LAMP assays and we have robust protocols for CSFV, PRRSV-1 and -2, PEDV, rotavirus A, as well as ASFV. Our Filipino collaborators have obtained follow-on funding to further optimise the ASFV RT-LAMP for application within the current outbreak and only recently got further funding to produce this test kit on a large scale to combat the ongoing ASFV outbreak in the country. The test kits using RT-LAMP analysis for the aforementioned viruses are also being developed for large-scale production and provision to Filipino farmers.
Exploitation Route Our methods developed in this project have broad applicability and we have obtained several bits of additional funding.
Diagnostics methods have been developed for RT-LAMP (we had to step away from RT-RPA due to difficulties obtaining reagents in the Philippines) and are made available to farmers and practitioners through the spin out company "Blitzkrieg" at cost.
The protocol for ASFV sequencing is being published on the Oxford Nanopore Technologies platform and Lilo will be made available on epi2me for use. We have already established several international partnerships and know of several labs starting to use the tiled sequencing for ASFV. This is likely going to be the new standard for sequencing of the virus.
We are expecting very similar impact for the PRRSV sequencing where we have a tiled whole-genome sequencing, an orf 2-7 (structural genes), and a single-read whole genome sequencing approach working.
Sectors Agriculture, Food and Drink,Government, Democracy and Justice,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description During this project together with our Filipino partners we have developed a variety of robust RT-LAMP assays for the diagnosis of porcine viral diseases. Some of these assays are now being made available to Filipino farmers and practitioners through a spin out company "Blitzkrieg" to diagnose and tackle viral disease. This has enabled the creation of local job opportunities and in particular, offers great opportunities for women.
Sector Agriculture, Food and Drink
Impact Types Societal,Economic

 
Description ASFv Nanogold
Amount ₱80,000,000 (PHP)
Organisation Department of Agriculture 
Sector Public
Country Philippines
Start 02/2021 
End 01/2024
 
Description Development of rapid diagnostics for ASFV by RT-LAMP (grant obtained by our collaborator on this project, Dr Clarissa Domingo)
Amount ₱11,000,000 (PHP)
Organisation Government of the Republic of the Philippines 
Sector Public
Country Philippines
Start 12/2019 
End 07/2021
 
Description Strengthening Animal Production and Health through the Immune Response (SAPHIR) - Sequencing of PRRSV samples
Amount € 10,549,121 (EUR)
Funding ID 633184 
Organisation European Commission H2020 
Sector Public
Country Belgium
Start 03/2015 
End 02/2019
 
Description [18-EEID US-UK DDCOVMP] Drivers of diversity and transmission of co-circulating viral lineages in host meta-populations
Amount £407,215 (GBP)
Funding ID BB/T004401/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2019 
End 08/2023
 
Title Lilo - tiled amplicon assembly pipeline 
Description Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly other methods fail on. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact Lilo provides a new amplicon polishing and assembly method that outperforms previously existing methods by enabling the assembly of highly variable and indel regions of tiled amplicon sequences. Originally developed for PRRSV assembly, where highly variable regions lead to failed assemblies on existing pipelines, such as ARTIC, Lilo continues to outperform other pipelines and has been integrated in our sequencing methods for ASFV but also performs exceptional on assembly of SARS-CoV-2. Lilo is going to be integrated into the Epi2me hub offered by Oxford Nanopore Technologies plc for wider public access with little background knowledge of bioinformatics. 
URL https://github.com/amandawarr/Lilo
 
Description Development of an optimised protocol for the sequencing of ASFV using ONT technology 
Organisation Oxford Nanopore Technologies
Country United Kingdom 
Sector Private 
PI Contribution We have developed primers, sequencing method and bioinformatics pipeline for the sequencing and assembly of whole genomes from African Swine Fever Virus (ASFV) samples for the purposes of research and epidemiological tracking of the virus. We used ONT sequencing for the broad applicability in ODA countries.
Collaborator Contribution ONT have provided us with in kind contributions of their sequencing equipment and funding towards staff to test and validate the protocols developed with samples from different countries and using different ASFV genotypes. They have brought us in contact with different international partners, which we hope to further develop into established collaborations.
Impact An ONT protocol is being finalised for publication on the Nanopore website following and referencing our preprint (10.1101/2021.12.01.470769v1?rss=1) soon to be submitted for publication. An international online seminar and Q&A on the use of this protocol and publicised and made available through the ONT website. Lilo, our assembly pipeline for tiled sequencing will be integrated within the Epi2me ONT platform for accessibility for people from ODA countries and people with limited bioinformatics background.
Start Year 2021
 
Description Exchange of ASFV DNA samples for sequencing 
Organisation Friedrich Loeffler Institute
Country Germany 
Sector Academic/University 
PI Contribution Sequencing of whole genome ASFV sequences from historic samples.
Collaborator Contribution Provision of ASFV DNA samples.
Impact ASFV sequences to aid epidemiological tracing and inform mutagenesis of the virus.
Start Year 2022
 
Description Exchange of ASFV DNA samples for sequencing 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Sequencing of whole genome ASFV sequences from historic samples.
Collaborator Contribution Provision of ASFV DNA samples.
Impact ASFV sequences to aid epidemiological tracing and inform mutagenesis of the virus.
Start Year 2021
 
Description Knowledge exchange and primers for ASFV sequencing 
Organisation National Academy of Sciences of the Republic of Armenia
Country Armenia 
Sector Academic/University 
PI Contribution Support with ASFV sequencing and analysis.
Collaborator Contribution ASFV samples and sequencing from current outbreaks.
Impact ASFV sequences to aid epidemiological tracing and inform mutagenesis of the virus.
Start Year 2022
 
Description Knowledge exchange and primers for ASFV sequencing 
Organisation Sokoine University of Agriculture
Country Tanzania, United Republic of 
Sector Academic/University 
PI Contribution Support with ASFV sequencing and analysis.
Collaborator Contribution Archived ASFV samples sequenced at location.
Impact ASFV sequences to aid epidemiological tracing and inform mutagenesis of the virus.
Start Year 2022
 
Description Knowledge exchange and primers for ASFV sequencing 
Organisation University of the Philippines Mindanao
Country Philippines 
Sector Academic/University 
PI Contribution Support with ASFV sequencing and analysis.
Collaborator Contribution ASFV samples and sequencing from current outbreaks.
Impact ASFV sequences to aid epidemiological tracing and inform mutagenesis of the virus.
Start Year 2021
 
Description CLSU "Updates on Swine Diagnostics" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact A presentation on the benefits of new pen-side diagnostics and how they could help decision making by farmers and veterinary professionals. The audience were Veterinary students from Central Luzon State University (The Philippines).
Year(s) Of Engagement Activity 2018
 
Description Invited talk European Veterinary Vaccinology Workshop 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Talk on career opportunities in academia for young scientists
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=hTujbImUCcI
 
Description Novel Control Strategies for Livestock and Poultry Diseases 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Our collaboration partner Clarisssa Y. Domingo co-organised and participated in a workshop in Manila, bringing together British and Filipino Veterinary practitioners, policy makers and scientists to discuss " Novel Control Strategies for Livestock and Poultry Diseases" and how improved diagnostics and epidemiological analysis can help with this process.
Year(s) Of Engagement Activity 2019
 
Description Panel interview for Chinese media 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Under the framework of the Newton Swine and Poultry Workshop in Beijing, China, a panel interview with selected grant holders of the "UK-China-Philippines-Thailand Swine and Poultry Research Initiative" was held at China Agricultrual University. Chinese and China-based international press was in attendance (amongst others, Reuters) and reported on the initiative, developing new antiviral strategies, diagnostics and on other developments in the pig- and poultry industry in China and related research.
Year(s) Of Engagement Activity 2018
 
Description Pirbright Institute Scientific Seminar - Tales of two viruses - Nanopore sequencing, genome editing, and stem cell models for ASFV and PRRSV 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk on genome editing for resistance, nanopore sequencing of ASFV and PRRSV. Outcome was the setting up of new collaborations and scientific input.
Year(s) Of Engagement Activity 2021
 
Description Seminar on genome editing for PRRSV resistance at CLSU, Munoz, The Philippines 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Genome editing for disease resistance in livestock in general and in pigs for PRRSV resistance was presented to all veterinary students and other department students at Central Luzon State University in the Philippines. Students could inquire the techniques and developments in this field and assess identify new approaches to disease treatments / prevention in animals.
Year(s) Of Engagement Activity 2019
 
Description The other deadly virus: tiled amplicon sequencing and assembly of African swine fever virus 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact African swine fever virus (ASFV) is the causative agent of a deadly, panzootic disease, infecting wild and domesticated suid populations. Contained for a long time to the African continent, an outbreak of a particularly infectious variant in Georgia in 2007 initiated the spread of the virus around the globe, severely impacting pork production and local economies. The virus is highly contagious and has a mortality of up to 100% in domestic pigs. It is critical to track the spread of the virus, detect variants associated with pathology, and implement biosecurity measures in the most effective way to limit its spread. Due to its size and other limitations, the 170-190 kbp large DNA virus genome has not been well sequenced, with fewer than 200 genome sequences available in public repositories. In this talk, we will present an efficient, low-cost method of sequencing ASFV at scale. The method uses tiled PCR amplification of the virus to achieve greater coverage, multiplexability and accuracy on a portable sequencer than is achievable using shotgun sequencing. We will also introduce Lilo, a pipeline for assembling tiled amplicon data from viral or microbial genomes without relying on polishing against a reference, allowing for structural variation and hypervariable region assembly, which other methods fail on. The ASFV genomes produced using this method are near complete, lacking only parts of the highly repetitive 3' and 5' telomeric regions, and have a high level of accuracy. These methods will allow sequencing of ASFV at optimal efficiency and high throughput to monitor and act on the spread of the virus as part of the global panzootic response.
Year(s) Of Engagement Activity 2022
URL https://nanoporetech.com/webinar/other-deadly-virus-tiled-amplicon-sequencing-and-assembly-african-s...