Recovery of viral RNA and infectious foot-and-mouth disease virus from positive lateral-flow devices

Foot-and-mouth disease Virus (FMDV) is an economically important, highly contagious picornavirus that affects both wild and domesticated cloven hooved animals. In developing countries, the effective laboratory diagnosis of foot-and-mouth disease (FMD) is often hindered by inadequate sample preservation due to difficulties in the transportation and storage of clinical material. These factors can compromise the ability to detect and characterise FMD virus in countries where the disease is endemic. Furthermore, the high cost of sending infectious virus material and the biosecurity risk it presents emphasises the need for a thermo-stable, non-infectious mode of transporting diagnostic samples. This paper investigates the potential of using FMDV lateral-flow devices (LFDs) for dry transportation of clinical samples for subsequent nucleic acid amplification, sequencing and recovery of infectious virus by electroporation. FMDV positive samples (epithelial suspensions and cell culture isolates) representing four FMDV serotypes were applied to antigen LFDs: after which it was possible to recover viral RNA that could be detected using real-time RT-PCR. Using this nucleic acid, it was also possible to recover VP1 sequences and also successfully utilise protocols for amplification of complete FMD virus genomes. It was not possible to recover infectious FMDV directly from the LFDs, however following electroporation into BHK-21 cells and subsequent cell passage, infectious virus could be recovered. Therefore, these results support the use of the antigen LFD for the dry, non-hazardous transportation of samples from FMD endemic countries to international reference laboratories

Reconstruction of the Transmission History of RNA Virus Outbreaks Using Full Genome Sequences: Foot-and-Mouth Disease Virus in Bulgaria in 2011

<div><p>Improvements to sequencing protocols and the development of computational phylogenetics have opened up opportunities to study the rapid evolution of RNA viruses in real time. In practical terms, these results can be combined with field data in order to reconstruct spatiotemporal scenarios that describe the origin and transmission pathways of viruses during an epidemic. In the case of notifiable diseases, such as foot-and-mouth disease (FMD), these analyses provide important insights into the epidemiology of field outbreaks that can support disease control programmes. This study reconstructs the origin and transmission history of the FMD outbreaks which occurred during 2011 in Burgas Province, Bulgaria, a country that had been previously FMD-free-without-vaccination since 1996. Nineteen full genome sequences (FGS) of FMD virus (FMDV) were generated and analysed, including eight representative viruses from all of the virus-positive outbreaks of the disease in the country and 11 closely-related contemporary viruses from countries in the region where FMD is endemic (Turkey and Israel). All Bulgarian sequences shared a single putative common ancestor which was closely related to the index case identified in wild boar. The closest relative from outside of Bulgaria was a FMDV collected during 2010 in Bursa (Anatolia, Turkey). Within Bulgaria, two discrete genetic clusters were detected that corresponded to two episodes of outbreaks that occurred during January and March-April 2011. The number of nucleotide substitutions that were present between, and within, these separate clusters provided evidence that undetected FMDV infection had occurred. These conclusions are supported by laboratory data that subsequently identified three additional FMDV-infected livestock premises by serosurveillance, as well as a number of antibody positive wild boar on both sides of the border with Turkish Thrace. This study highlights how FGS analysis can be used as an effective on-the-spot tool to support and help direct epidemiological investigations of field outbreaks.</p> </div

Box plot of Ct values generated from FMDV 3D rRT-PCR performed on sections of LFDs from serotype SAT 1 isolate (ZAM 5/2008) eluted at one month after LFD testing.

<p>LFDs had either been stored at room temperature (RoomT) (green bar) or 37°C (red bar). LP: Loading Pad; WS: Wicking strip; Nitrocellulose Ab Band (AbB). Dotted blue line represents baseline data (Ct values from LFDs washed on day one).</p

Foot-and-mouth disease virus samples used in the study.

<p>ES: Tongue epithelial suspension. CC: Cell culture supernatant.</p><p>Foot-and-mouth disease virus samples used in the study.</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of 14 separate LFDs and corresponding values generated for MagNA pure extracted RNA from the equivalent original epithelial suspensions in parallel.

<p>LP: Loading Pad; WS: Wicking strip; NB: Nitrocellulose below Ab Band (NB); Nitrocellulose Ab Band (AbB); Nitrocellulose above Ab band (NA). LFDs spanned four serotypes O (red dots) (LFDs TUR 8/1969, BAR 2/2008, KUW 2/2008, SAU 3/2008, ZAM 5/2008, HKN 10/2005, IRN 53/2006, UKG 7B/2007, A (blue dots) (LFDs BAR 4/2009, IRN 1/2008, KEN 8/2008, TUR 20/2006, IRN 36/2007), Asia 1 (grey dots) (IRN 15/2001) and SAT 1 (yellow dots) (ZAM 5/2008).</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of 5 separate LFDs and MagNA pure extracted RNA from the original epithelial suspensions and recorded by time of LFD testing.

<p>LP: Loading Pad; WS: Wicking strip; NB: Nitrocellulose below Ab Band (NB); Nitrocellulose Ab Band (AbB); Nitrocellulose above Ab band (NA). LFDs spanned two serotypes O (red dots) (LFDs HKN 10/2005, UKG 7B/2007) and A (blue dots) (LFDs TUR 20/2006, IRN53/2006, IRN 36/2007).</p

Visualization of the 24 overlapping PCR products representing the complete FMDV genome generated from the loading pad (LP) from LFD loaded with UKG 7B/2007.

<p>1.8% agarose gel stained with ethidium bromide (0.2 µg per ml). Lane 1, S-fragment of the 5′UTR, Lanes 2–22 L fragment, Lanes 23 and 24, poly A fragment.</p

Ct values generated from FMDV 3D rRT-PCR performed on sections of LFDs in dilution series from serotype O isolate (BAR 2/2008) compared against the visual detection on the Ag LFD.

<p>LP: Loading Pad; WS: Wicking strip; Nitrocellulose Ab Band (AbB)(+)  =  genome detected; (-)  =  No Ct.</p

Antigen (Ag) ELISA results from electroporated BHK cells passed once and twice on BTY cells.

<p>LP: Loading Pad; WS: Wicking strip; NC: Nitrocellulose. +: Obvious CPE; +/-: Suspected CPE: -: No CPE. O  =  O serotype detected, A  =  A serotype detected, Asia 1  =  Asia 1 serotype detected. LP/WS: Loading Pad combined with Wicking Strip; NC: Nitrocellulose. (1)  =  LFDs washed the same day. (2)  =  LFDs washed one week later.</p><p>Antigen (Ag) ELISA results from electroporated BHK cells passed once and twice on BTY cells.</p