Characterization of the O/ME-SA/Ind-2001d foot-and-mouth disease virus epidemic recorded in the Maghreb during 2014-2015.

The O/ME-SA/Ind-2001d has been the main foot-and-mouth disease virus (FMDV) lineage responsible for FMD epidemics outside the Indian subcontinent from 2013 to 2017. In 2014, outbreaks caused by this FMDV lineage were reported in Maghreb, where it was initially detected in Algeria and Tunisia and later in Morocco. This was the first incursion of an FMDV type O of exotic origin in the Maghreb region after 14 years of absence. In this study, we report analyses of both VP1 and whole-genome sequences (WGSs) generated from 22 isolates collected in Algeria and Tunisia between 2014 and 2015. All the WGSs analysed showed a minimum pairwise identity of 98.9% at the nucleotide level and 99% at the amino acid level (FMDV coding region). All Tunisian sequences shared a single putative common ancestor closely related to FMDV strains circulating in Libya during 2013. Whereas sequences from Algeria suggest the country experienced two virus introductions. The first introduction is represented by strains circulating in 2014 which are closely related to those from Tunisia, the second one, of which the origin is more uncertain, includes strains collected in Algeria in 2015 that gave origin to the 2015 outbreak reported in Morocco. Overall, our results demonstrated that a unique introduction of O/Ind-2001d FMDV occurred in Maghreb through Tunisia presumably in 2014, and from then the virus spread into Algeria and later into Morocco.

Safe and cost-effective protocol for shipment of samples from Foot-and-Mouth Disease suspected cases for laboratory diagnostic.

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.

Development of a Double-Antigen Microsphere Immunoassay for Simultaneous Group and Serotype Detection of Bluetongue Virus Antibodies.

Bluetongue viruses (BTV) are arboviruses responsible for infections in ruminants. The confirmation of BTV infections is based on rapid serological tests such as enzyme-linked immunosorbent assays (ELISAs) using the BTV viral protein 7 (VP7) as antigen. The determination of the BTV serotype by serological analyses could be only performed by neutralization tests (VNT) which are time-consuming and require BSL3 facilities. VP2 protein is considered the major serotype-defining protein of BTV. To improve the serological characterization of BTV infections, the recombinant VP7 and BTV serotype 8 (BTV-8) VP2 were synthesized using insect cells expression system. The purified antigens were covalently bound to fluorescent beads and then assayed with 822 characterized ruminant sera from BTV vaccinations or infections in a duplex microsphere immunoassay (MIA). The revelation step of this serological duplex assay was performed with biotinylated antigens instead of antispecies conjugates to use it on different ruminant species. The results demonstrated that MIA detected the anti-VP7 antibodies with a high specificity as well as a competitive ELISA approved for BTV diagnosis, with a better efficiency for the early detection of the anti-VP7 antibodies. The VP2 MIA results showed that this technology is also an alternative to VNT for BTV diagnosis. Comparisons between the VP2 MIA and VNT results showed that VNT detects the anti-VP2 antibodies in an early stage and that the VP2 MIA is as specific as VNT. This novel immunoassay provides a platform for developing multiplex assays, in which the presence of antibodies against multiple BTV serotypes can be detected simultaneously.

Genetic Characterization of Serotypes A and Asia-1 Foot-and-mouth Disease Viruses in Balochistan, Pakistan, in 2011.

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.

Establishment and validation of two duplex one-step real-time RT-PCR assays for diagnosis of foot-and-mouth disease.

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.

Encephalomyocarditis virus mortality in semi-wild bonobos (Pan panicus).

BACKGROUND: Fatal myocarditis from encephalomyocarditis virus (EMCV) infection has previously been identified in sporadic and epidemic forms in many species of captive non-human primates probably including one bonobo (Pan paniscus). METHODS: We investigated the deaths of two bonobos that were suspicious of EMCV using a combination of histopathology, immunohistochemistry and, for one of the two bonobos, reverse transcription PCR. RESULTS: Histopathological examination of heart tissue from the two bonobos showed changes characteristic of EMCV. Immunohistochemical studies confirmed the presence of EMCV antigen in heart tissue of both and in kidney and intestine of one of the bonobos. EMCV RNA was also isolated from the serum of the bonobo tested. CONCLUSION: Together, these findings confirm that EMCV was responsible for deaths of the two bonobos. Strict separation of bonobos in particular and captive primates in general from potential sources of EMCV contamination should be maintained to prevent mortality caused by EMCV.

[Encephalomyocarditis virus]. / Le virus de l'encéphalomyocardite.

Encephalomyocarditis virus (EMCV) is a cardiovirus of Picornaviridae family. It has a word wide distribution and affects a wide range of domestic and wild animal species mainly rodents, pigs and non-human primates. Depending on the virus strain and the infected host, the virus can induce myocarditis, reproductive failure, diabetes or nervous disorders. The importance of EMCV as a cause of disease in humans is unknown. However, its wide host range and its biological characteristics make this virus a potential zoonotic agent. Furthermore, several direct and indirect arguments, suggest that human infections cannot be discarded. This review summarises the current knowledge on the molecular biology, the pathogenicity and the zoonotic aspects of this virus.

Serological survey of encephalomyocarditis virus infection in pigs in France.

Samples of serum from 76 gilts, 1440 sows, 1473 piglets and 3093 finishing pigs from 96 farrow-to-finish herds were tested for antibodies to encephalomyocarditis virus (EMCV) in microtitre serum neutralisation tests employing two strains of virus, one associated with myocarditis and the other with reproductive failure. The total seroprevalence of EMCV infection was 2.48 per cent. There was no significant difference between the seroprevalence of the reproductive failure strain (1.6 per cent) and the myocardial strain (1.85 per cent). The seroprevalence was higher in the gilts (6.57 per cent) and sows (5.13 per cent) than in the piglets (1 per cent) and finishing pigs (1.84 per cent), and the highest titres were observed in the sows (1:540) and finishing pigs (1:640). In the gilts, the difference in seroprevalence between the reproductive failure strain (3.95 per cent) and the myocardial strain (5.33 per cent) was wider than in the other groups.

Characterization of a recombinant encephalomyocarditis virus expressing the enhanced green fluorescent protein.

A recombinant encephalomyocarditis virus (rEMCV2887A-egfp) expressing the enhanced green fluorescent protein (EGFP) was produced. The EGFP gene was inserted in frame within the leader protein coding sequence of a full-length cDNA clone of EMCV. RNA transcripts derived from the recombinant full-length cDNA were synthesized in vitro and transfected into BHK-21 cells. The recombinant transcript RNA remained infectious despite the insertion of EGFP as shown by cytopathic effects on BHK-21 cells and by propagation of the rescued virus. The replication kinetics in BHK-21 cells and the pathogenicity in mice of rEMCV2887A-egfp did not differ significantly from that of the parental virus. The recombinant virus was shown to produce fluorescence in infected cells after at least five passages in BHK-21 cells. However, a decrease of EGFP expression was observed following serial passages, and this was associated with the accumulation of deletion mutations within the EGFP gene. Nevertheless, using EGFP autofluorescence, infected cells were easily detected in the brain of mice infected with the first-passage recombinant virus. These data demonstrate that rEMCV2887A-egfp could be a useful tool to study virus dissemination and pathogenicity when used at low passages.

Development of an internal control for the detection of the African swine fever virus by PCR.

For the detection of African swine fever virus (ASFV) by polymerase chain reaction (PCR) in clinical samples, an internal control was constructed to identify false negative results in each reaction. The internal control was designed in such a way that the same primer pair was used to amplify the internal control and the target DNA which were differentiated by size. The lower detection limit was reached at about 30 internal control DNA copies and about 50 genomic ASFV DNA copies. The use of the internal control revealed that from a total of 12 uninfected samples, 10 samples contained inhibitors. After a one in two dilution, all ten of these samples amplified the internal control satisfactorily. From a total of 16 samples from pigs suspected of having ASF infection, nine contained inhibitors. After a one in two dilution, we observed internal control amplification and target DNA amplification for four samples.

Expression and characterization of part of hog cholera virus non-structural proteins.

In a preceding paper, the molecular cloning and partial nucleotide sequence of the Alfort strain of hog cholera virus (HCV) was described. To study the genetic organization of the 3'-end of the HCV genome, which encodes some of the non-structural proteins, a cDNA fragment (S2.20) of 849 nucleotides was subcloned into the bacterial expression vector pGEX-3X and expressed in Escherichia coli as a S2.20-glutathione-S-transferase fusion protein (S2.20-GST). This protein was used to produce HCV-specific monoclonal antibodies. Using Western immunoblotting, these antibodies could be used to identify a specific gene product of the HCV Alfort strain. Three proteins, with relative molecular weights of 76, 107 and 145 kDa, were detected. These proteins were also observed for eight other HCV strains. With the bovine viral diarrhoea virus (BVDV) NADL strain and the border disease virus (BDV) Aveyron strain, only one protein, with a relative molecular weight of 72 kDa, was detected. With the BVDV New York strain, two proteins, with relative molecular weights of 70 and 100 kDa, were recognized. The significance of these findings with respect to pestivirus genomic organization is discussed.

Partial sequencing of hog cholera virus Alfort strain genome and its comparison with other pestivirus strains.

After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined. This sequence was compared with homologous parts of previously published pestivirus genomes. The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67%. Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively). Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC. These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage. Genomic relationships among the pestiviruses are discussed.