Multiple invasions of O1 FMDV serotype into Israel revealed by genetic analysis of VP1 genes of Israeli's isolates from 1989 to 2007.

Foot-and-mouth disease virus (FMDV), one of the most dangerous viruses affecting cloven-hoofed animals, comprises seven serotypes that do not mutually cross-protect, with a total of about 80 subtypes. The Middle East is an FMD-endemic region, with repeated FMD outbreaks and In spite of its compulsory vaccination policy in Israel, outbreaks occur repeatedly. In order to compare the Israeli isolates, the complete viral VP1 genes of representative viruses isolated during the major outbreaks from 1989 to 2007 were sequenced and subjected to phylogenetic analysis, which showed that each outbreak was initiated by introduction of a new virus lineage and not by endemic and resident viruses. The differences between the nucleotide sequences of the viruses from the various outbreaks were too big to fit a model of outbreaks caused by endemic virus. Based on this approach, it was revealed that the 2002 outbreak was originated by viruses that circulated in the Arabian peninsula in 1997-1998.

The use of lumpy skin disease virus genome termini for detection and phylogenetic analysis.

During 2006 and 2007 there were two outbreaks of lumpy skin disease (LSD) in Israel. An LSD virus (LSDV)-specific PCR assay was developed that can detected specifically LSDV even though the number of tested LSDV isolates were limited. Full-length sheep pox and LSDV genome sequences were aligned to find non-homologous regions, which were then used for preparing specific primers, whose specificity was tested against several LSDV DNA isolates and the system could detect all the different isolates. Specificity was tested with sheep pox, ORF and other DNA viruses such as bovine herpes 4: the primers did not support amplification of the expected-size fragments, therefore the system appears to be a valuable tool for detecting specifically LSDV. The newly developed system was activated at the first report of a possible disease outbreak. It confirmed the clinical picture, and was introduced subsequently into routine diagnosis. Phylogenetic analyses of a 466-bp fragment next to the genome ends showed that this system can distinguish between: sheep pox, goat pox and LSD, and the results revealed that the Israeli isolates from 2006 and 2007 are in the same clad and essentially identical to Ismaeliya 1989, Nigeria 1996, Senegal 1997, Cameroon 1996, the Kenya NI-2490 isolate, and the South African LD virulent isolate. In contrast the original 1958 LW Neethling vaccine appeared to be in a separate clad, suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the viruses tested suggesting that the South African attenuated LW vaccine developed from a different ancestral origin that the rest of the tested viruses or during the process of attenuating the virus by succession of egg inoculations.

Cleavage of Akabane virus S RNA in the brain of infected ruminants.

Since 2002 there has been a rise in arthrogryposis/hydranencephaly incidence in Israel, caused by Akabane viruses (AKAV) and possibly by Aino viruses. In response to the outbreak, serological, molecular-diagnostic and research tools were developed. AKAV sequences were detected by real-time RT-PCR in the brain tissue of 2 out of 20 tested calves and lambs that suffered from hydranencephaly. When the S segments from the two infected calves were characterized, it was concluded that the S genome were cleaved. In order to localize the cleavage site, the 3' segment of the S genome was cloned, sequenced, and found to be 430 bases long, which indicates a cleavage site between nucleotides 430 and 431 of the S segment in the antigenome. This cleavage site was found to be specific and not a result of degradation processes. Analysis of the S segment RNA secondary structure revealed that the cleavage site was located on a loop structure. Furthermore, flunking the cleavage site there are stretches of 7 or 8 bases long that were part of a stem with low free energy, which could stabilize the loop, making it accessible to an, as yet, uncharacterized cleavage mechanism.

A real-time RT-quantative(q)PCR for the detection of bovine ephemeral fever virus.

A quantitative reverse-transcriptase real-time PCR assay, using TaqMan chemistry, for detecting bovine ephemeral virus (BEFV) is described. Available G gene sequences of viral RNA were aligned, and primers and probes were designed to recognize the virus. To quantitate the viruses, cDNA containing the real-time amplicon was prepared with a forward primer carrying the T7 promoter sequences. Run-off transcription from the T7 promoter amplicon template was used to prepare cRNA. Ten-fold dilutions of the run-off viral transcript were used as templates for the reaction in which they served as standards to quantitate unknown viral samples. By using this system it was shown that as few as 10-100 copies of a viral genome could be detected.

Inhibition of foot-and-mouth disease virus replication by small interfering RNA.

Foot-and-mouth disease, caused by foot-and-mouth disease virus (FMDV), is one of the most dangerous diseases of cloven-hoofed animals and is a constant threat to the dairy and beef industries in the Middle East and other regions of the world, despite intensive vaccination programmes. In this work, the ability of specific small interfering (si)RNAs to inhibit virus replication in BHK-21 cells was examined. By using bioinformatic computer programs, all FMDV sequences in public-domain databases were analysed. The analysis revealed three regions of at least 22 bp with 100 % identity in all FMDV entries. From these sequences, three specific siRNA molecules were prepared and used to test the ability of siRNAs to inhibit virus replication. By using real-time quantitative PCR to measure the amount of viral RNA in infected cells, it was shown that virus replication was inhibited in cells that were transfected with siRNAs. When viral titres were examined, 100 % inhibition of growth could be demonstrated in cells transfected with a mixture of all three anti-FMDV siRNAs, compared with control cells transfected with anti-LacZ siRNA.

Akabane virus in Israel: a new virus lineage.

This report describes the first molecular characterization of Akabane virus (AKAV) in Israel. The virus was recognized by real-time RT-PCR in extracts from Culicoides imicola insects trapped at the Volcani Center located in the center of Israel. This is also the first report on the use of real-time RT-PCR to identify the virus. The quantitative capability of this technique was applied, and it was calculated that the insect extract contains 1.5 x 10(5) copies of the genome segment S. Following amplification of the small (S) genome segment, its nucleotide sequence was determined to have 93.4% identity or greater with the S segment of other AKAV isolates. The deduced amino acid (aa) sequence of the combined nucleocapsid and the non-structural protein showed more than 96.6% identity. Phylogentic trees constructed using the combined deduced nucleocapsid and the non-structural protein aa sequences showed that the Israeli isolate forms a fourth cluster of AKAV, indicating a separate virus lineage. Attempts to isolate the virus by inoculation to Vero cells and by intracerebral inoculation to mice were unsuccessful.

Detection and quantitation of akabane and aino viruses by multiplex real-time reverse-transcriptase PCR.

A multiplex, quantitive reverse-transcriptase real-time PCR, using MGB TaqMan chemistry, for detecting akabane virus (AKAV) and aino virus (AINV) is described. Each specific probe was labeled with a different fluorescent dye--VIC for detecting AKAV and 6-carboxy-fluorescein (FAM) for detecting AINV. All available sequences of viral S RNA were aligned and primers and probes were designed so that AKAV primers and probes would recognize all AKAVs but not AINV, and vice versa. The parameters for multiplex reactions enabled the detection of both viruses in one tube reaction with similar efficiency. To quantitate the viruses, cDNA amplicons containing the real-time amplicon were prepared using forward primers carrying the T7 promoter sequences. The cDNAs were used directly as templates for run-off transcription and 10-fold dilutions of the products served as standards to quantitate unknown viral samples. Using this system had shown that it could detect approximately 3-30 copies of viral S genome.