Vesicular stomatitis – testing using reverse transcriptase loop-mediated isothermal amplification

• Vesicular Stomatitis New Jersey virus (VSNJV), in the family Rhabdoviridae, genus Vesiculovirus, is endemic in southern Mexico, Central America and northern regions of South America (Colombia, Venezuela, Ecuador and Peru).
• VSNJV is an arbovirus that can affect humans and causes vesicular stomatitis (VS) in horses, cattle and pigs.
• There have been a number of multiyear outbreaks of VSNJV in the United States which can cause heightened concern when the disease is observed in cattle or pigs due to the similarity in clinical presentation to Foot and mouth disease Foot and mouth disease (FMD).
• Loop-mediated isothermal amplification (LAMP) is an isothermal, autocycling, strand-displacement DNA amplification technique.
• LAMP utilises 4-6 primers which target 6-8 regions of the pathogen’s genome.
• LAMP can combine a reverse transcription step (RT-LAMP) to detect RNA viruses such as VSV.
• LAMP can be performed at a single temperature on a dedicated LAMP machine or be performed using a heat block/water bath combined with simple, disposable visualisation using molecular lateral-flow devices (LFD’s).
• LAMP is a highly sensitive technique that allows for the detection of a very low number of copies of RNA molecules with comparable analytical sensitivity to rRT-PCR.
• LAMP assays can be multiplexed to detect multiple pathogens in a single reaction.

Uses

Alone

• Within the literature there are increasing numbers of LAMP assays for the detection of veterinary pathogens.
• The most common application of LAMP is as a simple screening assay to rapidly confirm disease (<30 minutes) by detecting very low amounts of circulating pathogen nucleic acid (DNA or RNA).
• This, in combination with the development of portable LAMP platforms or visualisation of LAMP products on LFDs, enables disease confirmation at the point of suspicion (eg on the farm).
• Quick confirmation of disease may enable control measures to be implemented immediately.

In combination

• Field results generated from the use of LAMP assays at the point of suspicion, should be supported by laboratory diagnostics as recommended by the OIE: 
  • Virus isolation (VI).
  • Indirect sandwich ELISA.
  • Reverse transcriptase polymerase chain reaction.
  • Liquid-phase blocking ELISA.
  • Competitive ELISA.
  • Virus neutralisation test (VNT). 

Other points

• VS Stomatitis cannot reliably be clinically differentiated from the other vesicular diseases in the relevant susceptible species (cattle/pigs), such as FMD Foot and mouth disease.
• An early field/laboratory diagnosis of any suspected VS case is therefore a matter of urgency.
• LAMP can be performed in laboratory settings using automated nucleic acid extraction platforms, or can be performed in the field using manual extraction kits or by simply diluting the sample 1:5 in nuclease free water.
• LAMP assays are highly sensitive with comparable analytical sensitivity to real-time RT-PCR.
• LAMP assays are less prone to sample derived inhibition and can be performed on a wide range of clinical samples. Some LAMP assays, including the VSV LAMP assay has been validated for use without combining an extraction step.