Rapid Detection of Peste des Petits Ruminants Virus (PPRV) Nucleic Acid Using a Novel Low-Cost Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Assay for Future Use in Nascent PPR Eradication Programme.

Mahapatra Mahapatra,Howson Howson,Selvaraj Selvaraj,Flannery Flannery,Fowler Fowler,Batten Batten,Parida Parida

doi:10.3390/v11080699

Abstract

Peste des petits ruminants (PPR) is a disease of small ruminants caused by peste des petits ruminants virus (PPRV), and is endemic in Asia, the Middle East and Africa. Effective control combines the application of early warning systems, accurate laboratory diagnosis and reporting, animal movement restrictions, suitable vaccination and surveillance programs, and the coordination of all these measures by efficient veterinary services. Molecular assays, including conventional reverse transcription-polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR) have improved the sensitivity and rapidity of diagnosing PPR. However, currently these assays are only performed within laboratory settings; therefore, the development of field diagnostics for PPR would improve the fast implementation of control policies, particularly when PPR has been targeted to be eradicated by 2030. Loop-mediated isothermal amplification (LAMP) assays are simple to use, rapid, and have sensitivity and specificity within the range of RT-qPCR; and can be performed in the field using disposable consumables and portable equipment. This study describes the development of a novel RT-LAMP assay for the detection of PPRV nucleic acid by targeting the N-protein gene. The RT-LAMP assay was evaluated using cell culture propagated PPRVs, field samples from clinically infected animals and samples from experimentally infected animals encompassing all four lineages (I-IV) of PPRV. The test displayed 100% concordance with RT-qPCR when considering an RT-qPCR cut-off value of CT >40. Further, the RT-LAMP assay was evaluated using experimental and outbreak samples without prior RNA extraction making it more time and cost-effective. This assay provides a solution for a pen-side, rapid and inexpensive PPR diagnostic for use in the field in nascent PPR eradication programme.

Highlights

Peste des petits ruminants (PPR) is a highly contagious and economically important disease of small ruminants caused by PPR virus (PPRV) of the genus Morbillivirus in the family Paramyxoviridae
Optimisation of the RT-Loop-mediated isothermal amplification (LAMP) assay was conducted by examining four different primer concentrations, using RNA derived from PPRV/Morocco/2008 strain (Lineage IV)
Since the N-protein gene is expressed to a very high level in virus-infected cells, it has been used as an antigen for diagnostic purposes for rabies virus [24], measles virus [25], Newcastle disease virus (NDV) [26] and PPRV [23,27]

Summary

Introduction

Cattle and buffalo are sub-clinically infected; in addition, a large number of wild species within the order Artiodactyla are affected by PPRV [1]. PPR is endemic in large parts of Africa, the Middle East and Asia, and is still spreading globally, with emergence notably reported in China, Georgia, Mongolia, and most recently within the European Union in Bulgaria [1,2]. PPRV contains a single-stranded negative-sense RNA genome of about 16 kb, organised into six transcription units encoding six Viruses 2019, 11, 699; doi:10.3390/v11080699 www.mdpi.com/journal/viruses. Viruses 2019, 11, 699 structural proteins in the order 3’-N-P-M-F-H-L-5’. Two non-structural proteins, C and V are translated from the P-gene open reading frame (ORF) [3]. The eradication program would benefit from new diagnostic tools, field tests to enable detection of viral nucleic acid at the pen side

Methods

Results

Conclusion