Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

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Abstract

Rapid and accurate diagnosis of foot-and-mouth disease (FMD) and virus serotyping are of paramount importance for control of this disease in endemic areas where vaccination is practiced. Ideally this virus characterization should be achieved without the need for virus amplification in cell culture. Due to the heterogeneity of FMD viruses (FMDVs) in different parts of the world, region specific diagnostic tests are required. In this study, hydrolysable probe-based real time reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays were developed for specific detection and serotyping of the FMDVs currently circulating in West Eurasia. These assays were evaluated, in parallel with pan-FMDV diagnostic assays and earlier serotype-specific assays, using field samples originating from Pakistan and Afghanistan containing FMD viruses belonging to different sublineages of O-PanAsia, A-Iran05 and Asia-1 (Group-II and Group-VII (Sindh-08)). In addition, field samples from Iran and Bulgaria, containing FMDVs belonging to the O-PanAsia ^ANT-10 sublineage were also tested. Each of the three primer/probe sets was designed to be specific for just one of the serotypes O, A and Asia-1 of FMDV and detected the RNA from the target viruses with cycle threshold (C _T) values comparable with those obtained with the serotype-independent pan-FMDV diagnostic assays. No cross-reactivity was observed in these assays between the heterotypic viruses circulating in the region. The assays reported here have higher diagnostic sensitivity (100% each for serotypes O and Asia-1, and 92% [95% CI = 81.4–100%] for serotype A positive samples) and specificity (100% each for serotypes O, A and Asia-1 positive samples) for the viruses currently circulating in West Eurasia compared to the serotyping assays reported earlier. Comparisons of the sequences of the primers and probes used in these assays and the corresponding regions of the circulating viruses provided explanations for the poor recognition of some of the viruses by the earlier assays. These new assays should help in the early detection and typing of serotype O, A and Asia-1 FMDVs circulating in West Eurasia to enable improved disease control.

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The economic impacts of foot and mouth disease – What are they, how big are they and where do they occur?

T.J.D. Knight-Jones, J Rushton (2013)

Although a disease of low mortality, the global impact of foot and mouth disease (FMD) is colossal due to the huge numbers of animals affected. This impact can be separated into two components: (1) direct losses due to reduced production and changes in herd structure; and (2) indirect losses caused by costs of FMD control, poor access to markets and limited use of improved production technologies. This paper estimates that annual impact of FMD in terms of visible production losses and vaccination in endemic regions alone amount to between US$6.5 and 21 billion. In addition, outbreaks in FMD free countries and zones cause losses of >US$1.5 billion a year. FMD impacts are not the same throughout the world: 1. FMD production losses have a big impact on the world's poorest where more people are directly dependent on livestock. FMD reduces herd fertility leading to less efficient herd structures and discourages the use of FMD susceptible, high productivity breeds. Overall the direct losses limit livestock productivity affecting food security. 2. In countries with ongoing control programmes, FMD control and management creates large costs. These control programmes are often difficult to discontinue due to risks of new FMD incursion. 3. The presence, or even threat, of FMD prevents access to lucrative international markets. 4. In FMD free countries outbreaks occur periodically and the costs involved in regaining free status have been enormous. FMD is highly contagious and the actions of one farmer affect the risk of FMD occurring on other holdings; thus sizeable externalities are generated. Control therefore requires coordination within and between countries. These externalities imply that FMD control produces a significant amount of public goods, justifying the need for national and international public investment. Equipping poor countries with the tools needed to control FMD will involve the long term development of state veterinary services that in turn will deliver wider benefits to a nation including the control of other livestock diseases.

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Foot-and-mouth disease: past, present and future

Syed M. Jamal, Graham Belsham (2013)

Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals including cattle, pigs, sheep and many wildlife species. It can cause enormous economic losses when incursions occur into countries which are normally disease free. In addition, it has long-term effects within countries where the disease is endemic due to reduced animal productivity and the restrictions on international trade in animal products. The disease is caused by infection with foot-and-mouth disease virus (FMDV), a picornavirus. Seven different serotypes (and numerous variants) of FMDV have been identified. Some serotypes have a restricted geographical distribution, e.g. Asia-1, whereas others, notably serotype O, occur in many different regions. There is no cross-protection between serotypes and sometimes protection conferred by vaccines even of the same serotype can be limited. Thus it is important to characterize the viruses that are circulating if vaccination is being used for disease control. This review describes current methods for the detection and characterization of FMDVs. Sequence information is increasingly being used for identifying the source of outbreaks. In addition such information can be used to understand antigenic change within virus strains. The challenges and opportunities for improving the control of the disease within endemic settings, with a focus on Eurasia, are discussed, including the role of the FAO/EuFMD/OIE Progressive Control Pathway. Better control of the disease in endemic areas reduces the risk of incursions into disease-free regions.

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Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes).

N. Knowles, Olowookere Samuel (2001)

Serotype O is the most prevalent of the seven serotypes of foot-and-mouth disease (FMD) virus and occurs in many parts of the world. The UPGMA method was used to construct a phylogenetic tree based on nucleotide sequences at the 3' end of the VP1 gene from 105 FMD type O viruses obtained from samples submitted to the OIE/FAO World Reference Laboratory for FMD. This analysis identified eight major genotypes when a value of 15% nucleotide difference was used as a cut-off. The validity of these groupings was tested on the complete VP1 gene sequences of 23 of these viruses by bootstrap resampling and construction of a neighbour-joining tree. These eight genetic lineages fell within geographical boundaries and we have used the term topotype to describe them. Using a large sequence database, the distribution of viruses belonging to each of the eight topotypes has been determined. These phylogenetically based epidemiological studies have also been used to identify viruses that have transgressed their normal ecological niches. Despite the high rate of mutation during replication of the FMD virus genome, the topotypes appear to represent evolutionary cul-de-sacs.

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Author and article information

Contributors

Nagendra R Hegde: Role: Editor

Journal

Journal ID (nlm-ta): PLoS One

Journal ID (iso-abbrev): PLoS ONE

Journal ID (publisher-id): plos

Journal ID (pmc): plosone

Title: PLoS ONE

Publisher: Public Library of Science (San Francisco, CA USA )

ISSN (Electronic): 1932-6203

Publication date (Electronic): 13 August 2015

Publication date Collection: 2015

Volume: 10

Issue: 8

Electronic Location Identifier: e0135559

Affiliations

[1 ]Department of Biotechnology, University of Malakand, Chakdara, Dir (L), Khyber Pakhtunkhwa, Pakistan

[2 ]National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark

Ella Foundation, INDIA

Author notes

Competing Interests: The authors have declared that no competing interests exist.

Conceived and designed the experiments: SMJ GJB. Performed the experiments: SMJ. Analyzed the data: SMJ GJB. Wrote the paper: SMJ GJB.

* E-mail: grbe@ 123456vet.dtu.dk

Article

Publisher ID: PONE-D-15-15196

DOI: 10.1371/journal.pone.0135559

PMC ID: 4535879

PubMed ID: 26270532

SO-VID: f4b04fad-4c01-4807-a0c8-8273c08356d1

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This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

History

Date received : 8 April 2015

Date accepted : 23 July 2015

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Figures: 6, Tables: 2, Pages: 16

Funding

The work was funded internally within the DTU (Technical University of Denmark) National Veterinary Institute ( www.vet.dtu.dk) and in part by the European Commission for the Control of Foot-and-mouth disease (EuFMD)( http://www.fao.org/ag/againfo/commissions/eufmd/commissions/eufmd-home/en/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Development and Characterization of Probe-Based Real Time Quantitative RT-PCR Assays for Detection and Serotyping of Foot-And-Mouth Disease Viruses Circulating in West Eurasia

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Most cited references 31

The economic impacts of foot and mouth disease – What are they, how big are they and where do they occur?

Foot-and-mouth disease: past, present and future

Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes).

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