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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.

RNA-mediated interference (RNAi) is a recently discovered process by which dsRNA is able to silence specific gene functions. Although initially described in plants, nematodes and Drosophila, the process is currently considered to be an evolutionarily conserved process that is present in the entire eukaryotic kingdom in which its original function was as a defense mechanism against viruses and foreign nucleic acids. Similarly to the silencing of genes by RNAi, viral functions can be also silenced by the same mechanism, through the introduction of specific dsRNA molecules into cells, where they are targeted to essential genes or directly to the viral genome in case RNA viruses, thus arresting viral replication. Since the pioneering work of Elbashir and coworkers, who identified RNAi activity in mammalian cells, many publications have described the inhibition of viruses belonging to most if not all viral families, by targeting and silencing diverse viral genes as well as cell genes that are essential for virus replication. Moreover, virus expression vectors were developed and used as vehicles with which to deliver siRNAs into cells. This review will describe the use of RNAi to inhibit virus replication directly, as well as through the silencing of the appropriate cell functions.

The A genome segment of the highly virulent Infectious bursal disease virus (IBDV) was amplified using long and accurate-RT-PCR (LA-RT-PCR). The entire sequence region encoding VP2, VP4, and VP3 in that order was cloned and sequenced. Following subcloning into the Escherichia coli expression vector pET21a under the T7 promoter, viral proteins were expressed and processed as demonstrated by Western blot analysis. Virus-like particles could be visualized by immuno-electron microscopy in IPTG-induced cells suggesting that viral assembly can take place in E. coli. Induction of anti-IBDV antibodies was detected in chickens immunized with purified recombinant IBDV by intra muscular (i.m.) injection. Furthermore, the vaccinated chickens were protected when challenged with the Gep 5 isolate of IBDV.

In November 1997, an outbreak of a neuroparalytic disease caused by West Nile (WN) virus was diagnosed in young goose flocks. Domestic geese were similarly affected in the late summer and fall of 1998, 1999, 2000 and 2001. WN viruses were also isolated from migratory and wild birds and horses in 1998-2001. A 1278 bp sequence of the envelope gene of 24 Israeli WN virus isolates was compared with those of seven isolates from Africa, Europe and New York. As a result, the Israeli isolates could then be grouped into two clusters. The 15 avian and three equine from 1997-2001 in the first cluster of viruses were shown to be identical to WN-NY99, while the second cluster comprised one goose isolate from 1998 and two goose and two pigeon isolates from 2000. These closely resembled the most recent Old World isolates, and indicate that at least two WN genotypes were co-circulating in the region during this time.

The replication of foot and mouth disease virus (FMDV) was studied in isolated bovine skin Langerhans cells (LC), in keratinocytes from epidermal cell suspension, and in migrating LC obtained from cultured bovine epidermal sheets in vitro. Viral RNA replication in infected cells was determined by the reverse transcriptase-polymerase chain reaction (RT-PCR) of the negative FMDV RNA strand and by the plaque forming assay of FMDV. It was established that bovine skin LC, keratinocytes, and migratory bovine LC infected with FMDV strain 01 Geshur supported virus replication. This RT-PCR method to detect the negative strand of FMDV RNA in migratory bovine skin LC may be useful for determining FMD virus replication in tissue cells.

Two ribozyme genes were designed to cut within the VP4 and VP3 sequences of foot and mouth disease virus (FMDV) Asia1 serotype genome. The two genes were synthesized and cloned into pBluescript under the control of the T3 promoter. The ribozyme designed to cut the VP4 gene contained two catalytic sequences targeted to two GUC triplets that are 16 bases apart. The second ribozyme, intended to cut VP3, contained one catalytic sequence. Ribozymes obtained from run-off transcription from both plasmids were able to cleave viral RNA derived from runoff transcripts of plasmids carrying the proper FMDV cDNA inserts. The significance of these findings is discussed.

Differences in the amino acid sequence of foot-and-mouth disease virus (FMDV) virion proteins (VP) among the various FMDV serotypes, particularly in the VP1 polypeptide, are the basis for antigenic diversity of this virus group. This phenomenon provides the basis for type diagnosis of FMDV by the polymerase chain reaction (PCR). In order to specifically identify the Asia1 FMDV serotype by PCR, the nucleotide sequence of its P1-coding region was determined. The sequence exhibited over 70% homology with the P1 gene segment of type O1k. The deduced amino acid sequence shares 79% homology with that of the P1 region of serotype O1k.

Transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (HC) that also exhibits protease activity. HC was expressed in E. coli from two types of clones: a full-length cDNA clone of PVY and two 5' end clones containing the first three cistrons (3.6-3.7 kbp). The clones derived from the 5' end of PVY expressed HC of the size of the mature component. Other proteins reacting with antibodies to HC were also observed, and their sizes corresponded with those of expected intermediates resulting from partial protease cleavage of the three-cistron polyprotein. On the other hand, the only detectable HC-related product of the full-length clone was a mature-size HC. The presence of a third PVY protease among the first three cistrons is therefore suggested.

Full-length cDNA of genomic RNA of potato virus Y (PVY) was cloned in one piece into a lambda vector. The order of the EcoRI and SalI fragments of the inserted cDNA was determined. This is the first report of the cloning of a long, expressible, potyvirus genome. The availability of such a clone is a prerequisite for any further study of the molecular biology of this group of viruses, as they are expressed into a self-processed primary polyprotein.

Several reports have indicated that tobacco carries an enzyme (APE) that, in the presence of poly (rI):(rC), polymerizes ATP to oligoadenylates. This paper demonstrates that the tobacco APE system comprises several proteins (estimated sizes: 32, 42, 67, and 84 +/- 10% kD). Only one of these proteins (the "67-kD" form) binds to poly (rI):(rC). This APE form has been purified by affinity chromatography on a synthetic ds-RNA column. Four tobacco proteins, including the purified one, crossreact with antibodies against the human enzyme, 2'-5' A synthetase. The ATP-binding capacity of some of these proteins has also been demonstrated. The amount of plant oligoadenylates obtained by polymerizing ATP with the purified APE form allows, for the first time, their direct analysis by TLC. The TLC analysis indicated that the oligomer produced by APE is not identical to the 2'-5' oligoadenylate. The appearance of the 2'-5' A-related proteins correlates with the build up of TMV infection, and the pattern of their stimulation and turnover was established. Nucleic acid hybridization indicates homology of tobacco DNA and RNA sequences with cloned cDNA of the human 2'-5' A synthetase gene. The stimulation in tobacco, upon TMV infection, of mRNA species homologous to the above human cDNA has been demonstrated. The analogy between the plant and the human system is discussed.