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

Understanding foot-and-mouth disease virus transmission biology: identification of the indicators of infectiousness

The control of foot-and-mouth disease virus (FMDV) outbreaks in non-endemic countries relies on the rapid detection and removal of infected animals. In this paper we use the observed relationship between the onset of clinical signs and direct contact transmission of FMDV to identify predictors for the onset of clinical signs and identify possible approaches to preclinical screening in the field. Threshold levels for various virological and immunological variables were determined using Receiver Operating Characteristic (ROC) curve analysis and then tested using generalized linear mixed models to determine their ability to predict the onset of clinical signs. In addition, concordance statistics between qualitative real time PCR test results and virus isolation results were evaluated. For the majority of animals (71%), the onset of clinical signs occurred 3–4 days post infection. The onset of clinical signs was associated with high levels of virus in the blood, oropharyngeal fluid and nasal fluid. Virus is first detectable in the oropharyngeal fluid, but detection of virus in the blood and nasal fluid may also be good candidates for preclinical indicators. Detection of virus in the air was also significantly associated with transmission. This study is the first to identify statistically significant indicators of infectiousness for FMDV at defined time periods during disease progression in a natural host species. Identifying factors associated with infectiousness will advance our understanding of transmission mechanisms and refine intra-herd and inter-herd disease transmission models

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 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

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

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

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

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