ABSTRACT
An essential step towards the global control and eradication of foot-and-mouth disease (FMD) is the identification of circulating virus strains in endemic regions to implement adequate outbreak control measures. However, due to the high biological risk and the requirement for biological samples to be shipped frozen, the cost of shipping samples becomes one of major obstacles hindering submission of suspected samples to reference laboratories for virus identification. In this study, we report the development of a cost-effective and safe method for shipment of FMD samples. The protocol is based on the inactivation of FMD virus (FMDV) on lateral flow device (LFD, penside test routinely used in the field for rapid immunodetection of FMDV), allowing its subsequent detection and typing by RT-PCR and recovery of live virus upon RNA transfection into permissive cells. After live FMDV collection onto LFD strip and soaking in 0.2% citric acid solution, the virus is totally inactivated. Viral RNA is still detectable by real-time RT-PCR following inactivation, and the virus strain can be characterized by sequencing of the VP1 coding region. In addition, live virus can be rescued by transfecting RNA extract from treated LFD into cells. This protocol should help promoting submission of FMD suspected samples to reference laboratories (by reducing the cost of sample shipping) and thus characterization of FMDV strains circulating in endemic regions.
Subject(s)
Cost-Benefit Analysis , Foot-and-Mouth Disease Virus/isolation & purification , Foot-and-Mouth Disease/diagnosis , Foot-and-Mouth Disease/virology , Specimen Handling/economics , Animals , Cattle , Clinical Laboratory Techniques , Clinical Protocols , Foot-and-Mouth Disease Virus/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Safety Management , Sensitivity and Specificity , Specimen Handling/methods , Swine , Transfection , Virus InactivationABSTRACT
This study reports characterization of foot-and-mouth disease virus (FMDV) in samples collected from Balochistan, Pakistan. FMDV was detected by pan-FMDV real-time RT-PCR in 31 samples (epithelial and oral swabs) collected in 2011 from clinical suspect cases. Of these, 29 samples were serotyped by serotype-specific real-time RT-PCR assays and were confirmed by sequencing the VP1 coding region. Sixteen samples were found positive for serotype A and eight for serotype Asia-1, whereas five samples were found positive for both serotypes A and Asia-1. Two serotype A positive samples were found positive for two different strains of serotype A FMDV each. Phylogenetic analyses of serotype A FMDVs showed circulation of at least three different sublineages within the A-Iran05 lineage. These included two earlier reported sublineages, A-Iran05HER-10 and A-Iran05FAR-11 , and a new sublineage, designated here as A-Iran05BAL-11 . This shows that viruses belonging to the A-Iran05 lineage are continuously evolving in the region. Viruses belonging to the A-Iran05FAR-11 sublineage showed close identity with the viruses circulating in 2009 in Pakistan and Afghanistan. However, viruses belonging to the A-Iran05HER-10 detected in Balochistan, Pakistan, showed close identity with the viruses circulating in Kyrgyzstan, Iran and Kazakhstan in 2011 and 2012, showing that viruses responsible for outbreak in these countries have a common origin. Serotype Asia-1 FMDVs reported in this study all belonged to the earlier reported Group-VII (Sindh-08), which is currently a dominant strain in the West Eurasian region. Detection of two different serotypes of FMDV or/and two different strains of the same serotype in one animal/sample shows complexity in occurrence of FMD in the region.
Subject(s)
Disease Outbreaks , Foot-and-Mouth Disease Virus/classification , Foot-and-Mouth Disease/epidemiology , Animals , Foot-and-Mouth Disease Virus/genetics , Livestock , Pakistan/epidemiology , Phylogeny , Real-Time Polymerase Chain Reaction/methods , SerogroupABSTRACT
At the end of August 2015, a ram located in central France (department of Allier) showed clinical signs suggestive of BTV (Bluetongue virus) infection. However, none of the other animals located in the herd showed any signs of the Bluetongue disease. Laboratory analyses identified the virus as BTV serotype 8. The viro and sero prevalence intraherd were 2.4% and 8.6% in sheep and 18.3% and 42.9% in cattle, respectively. Phylogenetic studies showed that the sequences of this strain are closely related to another BTV-8 strain that has circulated in France in 2006-2008. The origin of the outbreak is unclear but it may be assumed that the BTV-8 has probably circulated at very low prevalence (possibly in livestock or wildlife) since its first emergence in 2007-2008.
Subject(s)
Bluetongue virus/classification , Bluetongue/virology , Cattle Diseases/virology , Communicable Diseases, Emerging/veterinary , Animals , Bluetongue/epidemiology , Cattle , Cattle Diseases/epidemiology , Chick Embryo , Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/virology , Cricetinae , Disease Outbreaks/veterinary , France/epidemiology , Male , Mice , Mice, Knockout , Phylogeny , Prevalence , Serogroup , SheepABSTRACT
Two duplex one-step TaqMan-based RT-PCR protocols for detection of foot-and-mouth disease virus (FMDV) were established and validated. Each RT-PCR test consists of a ready-to-use master mix for simultaneous detection of the well established 3D or IRES FMDV targets and incorporates the host ß-actin mRNA as an internal control target, in a single-tube assay. The two real-time RT-PCR 3D/ß-actin and IRES/ß-actin tests are highly sensitive and able to detect up to 7TCID50/ml of FMDV and 10 copies/1µl of viral RNA. In field epithelium samples, the diagnostic sensitivity was 100% (95% CI; 91-100%) for the 3D/ß-actin test and 97% (95% CI; 87-100%) for the IRES/ß-actin test. The diagnostic specificity was 100% (95% CI; 95-100%) for both RT-PCRs. In addition, the two protocols proved to be robust, showing inter-assay coefficients of variation ranging from 1.94% to 6.73% for the IRES target and from 2.33% to 5.42% for the 3D target for different RNA extractions and different RT-PCR conditions. The internally controlled one-step real-time RT-PCR protocols described in this study provide a rapid, effective and reliable method for the detection of FMDV and thus may improve the routine diagnosis for foot-and-mouth disease.
Subject(s)
Foot-and-Mouth Disease Virus/isolation & purification , Foot-and-Mouth Disease/diagnosis , Real-Time Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain Reaction/methods , Actins/genetics , Animals , Cattle , Cattle Diseases/diagnosis , Cattle Diseases/virology , Foot-and-Mouth Disease/virology , Foot-and-Mouth Disease Virus/genetics , Goat Diseases/diagnosis , Goat Diseases/virology , Goats , RNA, Viral/genetics , Sensitivity and Specificity , Sheep , Sheep Diseases/diagnosis , Sheep Diseases/virology , Tongue/cytology , Tongue/virologyABSTRACT
The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.
