Stretching the wings further- susceptibility of Culex pipiens Linnaeus to bovine ephemeral fever virus infection under experimental conditions.

Bovine ephemeral fever (BEF) is a significant viral disease of cattle in the tropical, subtropical, and temperate climatic zones. This disease is also known as three-day sickness due to the spontaneous recovery of the cattle within a short period (usually 3-5 days). Despite its short duration, the disease may have a considerable impact. It can cause heavy economic losses, primarily due to decreased milk production, lowered fertility in bulls, and even fatality in severe cases. The virus is suspected to be transmitted by haematophagous insects (mainly mosquitoes and Culicoides biting midges); however, the identity of a competent vector for BEFV remains a mystery. Here, we investigated whether BEFV may replicate for a short duration in Culex pipiens Linnaeus, 1758, the most prevalent mosquito species in Israel and a potential vector of this virus to Israeli cattle. We applied nested- qPCR to test BEFV abundance in Cx. pipiens every 24 h for 14 consecutive days post-infection. Additionally, we collected eggs laid by BEFV-infected females and investigated BEFV abundance in the different developmental stages of F1 mosquitos. Our results suggest that Cx. pipiens mosquitoes have the potential to act as a vector of BEFV and also indicate that BEFV may be vertically transmitted from Cx. pipiens female parent to her female offspring.

Importance of the lumpy skin disease virus (LSDV) LSDV126 gene in differential diagnosis and epidemiology and its possible involvement in attenuation.

Examination of lumpy skin disease virus (LSDV) isolates from different geographic regions and times revealed that assays developed in our laboratory for differentiating between virulent Israeli viruses and Neethling vaccine virus (NVV) are generally useful in most, if not all, endemic areas in which NVV-based vaccines are used. Recently it was revealed that the LSDV126 gene of field isolates contains a duplicated region of 27 bp (9 aa), while the vaccine viruses have only one copy. Phylogenetic analysis of a 532-bp segment carrying the LSDV126 gene and whole virus genome sequences revealed that LSDV isolates formed two groups: virulent and vaccine viruses. In this analysis, all of the capripox viruses that lack the ability to efficiently infect cattle were found to carry only one copy of the 27-bp fragment, suggesting that the LSDV126 gene plays an important role in the ability of capripox viruses to infect cattle. In silico analysis of potential antigenic sites in LSDV126 revealed that LSDV126 variants with only one copy of the repeat lack a potentially important antigenic epitope, supporting its possible significance in cattle infection. This study provides new information about the nature of the LSDV126 gene and its possible role in the life cycle of LSDV.

Pathological and molecular characterisation of peste des petits ruminants in Nubian ibex (Capra nubiana) in Israel.

Peste des petits ruminants (PPR) is a devastating disease that generally affects sheep and goats, mostly in Asia, the Middle East and Africa. The disease has been declared a target for global eradication. Despite its high prevalence in domestic flocks and its high seroprevalence among wildlife, it is rarely reported as a fulminant disease in wild ruminant species (with the exception of Central Asia). In this report, we describe a severe PPR outbreak in a zoo herd of Nubian ibex (Capra nubiana), causing the deaths of 2/3 of the herd. The clinical onset was acute with morbid animals exhibiting lethargy and watery-to-bloody diarrhea and death usually within 48 h. The most consistent gross pathologic findings were hemorrhagic abomasitis and enteritis. Oral lesions and pulmonary lesions were rare. Histology revealed necrohemorrhagic enteritis and abomasitis with myriad nuclear and cytoplasmic viral inclusion bodies. Molecular examinations confirmed the diagnosis of PPR and determined that the causative agent belongs to lineage IV. Further molecular examination showed that the virus belongs to the Asian clade of lineage IV and is closely related to a virus described in Turkey.

High-resolution melting (HRM) for genotyping bovine ephemeral fever virus (BEFV).

In recent years there have been several major outbreaks of bovine ephemeral disease in the Middle East, including Israel. Such occurrences raise the need for quick identification of the viruses responsible for the outbreaks, in order to rapidly identify the entry of viruses that do not belong to the Middle-East BEFV lineage. This challenge was met by the development of a high-resolution melt (HRM) assay. The assay is based on the viral G gene sequence and generation of an algorithm that calculates and evaluates the GC content of various fragments. The algorithm was designed to scan 50- to 200-base-long segments in a sliding-window manner, compare and rank them using an Order of Technique of Preference by Similarity to Ideal Solution (TOPSIS) the technique for order preference by similarity to ideal solution technique, according to the differences in GC content of homologous fragments. Two fragments were selected, based on a match to the analysis criteria, in terms of size and GC content. These fragments were successfully used in the analysis to differentiate between different virus lineages, thus facilitating assignment of the viruses' geographical origins. Moreover, the assay could be used for differentiating infected from vaccinated animales (DIVA). The new algorithm may therefore be useful for development of improved genotyping studies for other viruses and possibly other microorganisms.

What can Akabane disease teach us about other arboviral diseases.

Viruses of the Simbu serogroup cause lesions to foetuses that are seen at birth and that correlate with the stage of pregnancy at which the dam first contracts the virus. The Simbu serogroup comprises arboviruses known to cause outbreaks of abnormal parturitions in domestic ruminants; these abnormalities include abortion, stillbirth, and congenitally deformed neonates. Simbu serogroup members include: Akabane virus (AKAV), Aino virus, Cache Valley virus, and Schmallenberg virus. Lately, dairy herds calf malformations have been observed in Europe, where there have been reports of clinical manifestations such as diarrhoea, fever, and reduced milk yield in adult lactating cows. The Israeli dairy cattle industry has experienced 2 major episodes of abnormal parturitions that resulted from 2 arboviral Simbu serogroup episodes, which occurred 35 years apart. A wave of apparently newly introduced AKAV was noted from the beginning of January 2012. Investigations carried out throughout the period of late Summer 2011 to early Winter 2012, associated the Israeli AKAV strain with central nervous system manifestations in lactating cows. A lack of clinical/epidemiological 'uniformity' among the AKAV infections was noted during these investigations. Here we describe and discuss the clinical and spatial distribution differences found among the 3 above-mentioned outbreaks. Comparable features in the clinical presentation, spatial distribution, and target­animal issues relating to Akabane disease are discussed.

A high-resolution melting (HRM) assay for the differentiation between Israeli field and Neethling vaccine lumpy skin disease viruses.

Lumpy skin disease (LSD) is a constant threat to the Middle East including the State of Israel. During vaccination programs it is essential for veterinary services and farmers to be able to distinguish between animals affected by the cattle-borne virulent viruses and vaccinated animals, subsequently affected by the vaccine strain. This study describes an improved high resolution-melting (HRM) test that exploits a 27 base pair (bp) fragment of the LSDV126 extracellular enveloped virion (EEV) gene that is present in field viruses but is absent from the Neethling vaccine strain. This difference leads to ∼0.5 °C melting point change in the HRM assay, when testing the quantitative PCR (qPCR) products generated from the virulent field viruses compared to the attenuated vaccine. By exploiting this difference, it could be shown using the newly developed HRM assay that virus isolated from vaccinated cattle that developed disease symptoms behave similarly to vaccine virus control, indicating that the vaccine virus can induce disease symptoms. This assay is not only in full agreement with the previously published PCR gradient and restriction fragment length polymorphism (RFLP) tests but it is faster with, fewer steps, cheaper and dependable.

Genotyping and phylogenetic analysis of bovine viral diarrhea virus (BVDV) isolates in Kosovo.

Three serum samples positive in Antigen ELISA BVDV have been tested to characterise genetic diversity of bovine viral diarrhea virus (BVDV) in Kosovo. Samples were obtained in 2011 from heifers and were amplified by reverse transcription-polymerase chain reaction, sequenced and analysed by computer-assisted phylogenetic analysis. Amplified products and nucleotide sequence showed that all 3 isolates belonged to BVDV 1 genotype and 1b sub genotype. These results enrich the extant knowledge of BVDV and represent the first documented data about Kosovo BVDV isolates.

Development of an assay to differentiate between virulent and vaccine strains of lumpy skin disease virus (LSDV).

Lumpy skin disease (LSD) was and still is a constant threat to the State of Israel, since the first outbreaks in 1989 and in 2006-2007. Recently, another massive outbreak occurred, at the beginning of July 2012, in the northern part of Israel. An intensive vaccination campaign with a sheeppox-based vaccine was initiated, in addition to culling symptomatic animals in the dairy herds. In spite of this, there was a need to apply extra efforts to completely contain and control the spread of the disease by introducing for the first time in Israel a vaccine based on the Neethling vaccine virus strain. However, in case of appearance of LSD symptoms it was essential to be able to distinguish between cattle-carried virulent strain and the vaccine strain. This paper describes the development and utilization of a molecular assay that can differentiate between the virulent isolates from the vaccine strain. The system is based on 3 different tests; it was found that the vaccine strain carries 27 bases less than the virulent virus in the extracellular enveloped virions (EEV) gene. A temperature-gradient PCRs were done using primers which are identical to the vaccine strain but differ at the 3' end nucleotides to the virulent virus. PCR-RFLP was carried out on the presence of an MboI site unique to the vaccine strain. Thus, all three tests presented here are able to differentiate specifically between the two viral appearances.

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.

Epidemiological investigation of bovine ephemeral Fever outbreaks in Israel.

Outbreaks of bovine ephemeral fever (BEF) occurred in Israel in 1990, 1999, and 2004. The main patterns of BEF spread were similar in the 1990 and in 1999 epidemics, and the BEF virus was probably carried in vectors transported by air streams across the Rift Valley and the Red Sea. In the 2004 outbreak, the primary focus of the disease was the southern Mediterranean coastal plain and the disease agent was apparently brought by infected mosquitoes carried from their breeding site in the Nile Delta by the south-western winds. The disease broke out under optimal ecological conditions, among a vulnerable cattle population and spread rapidly; it showed essentially a spring-summer herd incidence and terminated soon after the night average ambient temperature fell below 16 degrees C in late autumn. The herd incidence of the disease reached 78.4%, 97.7%, and 100% in 1990, 1999, and 2004, respectively. The highest herd incidence, morbidity, and case fatality rates were noted in dairy cattle herds in the Jordan Valley, with morbidity of 20%, 38.6%, and 22.2%, and case fatality rate among affected animals of 2%, 8.6%, and 5.4% in 1990, 1999, and 2004, respectively. The average sero-positivity to BEF in 1999 was 39.5%, which matched the morbidity rate. Comparison among the various age groups showed that the lowest morbidity rates were observed in the youngest age group, that is, heifers up to 1 year, with 3.2%, 3.6%, and 4.2% in 1990, 1999, and 2004, respectively. In heifers from 1 year to calving, the morbidity rates were 13.8%, 14.9%, and 28%, respectively, in first calvers 30.8%, 31.6%, and 28.3%, respectively, and in cows 34.3%, 35.7%, and 27.2%, respectively. All affected cattle were over the age of 3 months. It is hypothesized that mosquitoes and not Culicoides spp. are the vectors of the BEF virus in Israel.

Use of sperm plasmid DNA lipofection combined with REMI (restriction enzyme-mediated insertion) for production of transgenic chickens expressing eGFP (enhanced green fluorescent protein) or human follicle-stimulating hormone.

Linearized p-eGFP (plasmid-enhanced green fluorescent protein) or p-hFSH (plasmid human FSH) sequences with the corresponding restriction enzyme were lipofected into sperm genomic DNA. Sperm transfected with p-eGFP were used for artificial insemination in hens, and in 17 out of 19 of the resultant chicks, the exogenous DNA was detected in their lymphocytes as determined by PCR and expressed in tissues as determined by (a) PCR, (b) specific emission of green fluorescence by the eGFP, and (c) Southern blot analysis. A complete homology was found between the Aequorea Victoria eGFP DNA and a 313-bp PCR product of extracted DNA from chick blood cells. Following insemination with sperm lipofected with p-hFSH, transgenic offspring were obtained for two generations as determined by detection of the transgene for human FSH (PCR) and expression of the gene (RT-PCR and quantitative real-time PCR) and the presence of the protein in blood (radioimmunoassay). Data demonstrate that lipofection of plasmid DNA with restriction enzyme is a highly efficient method for the production of transfected sperm to produce transgenic offspring by direct artificial insemination.

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.

Inhibition of viruses by RNA interference.

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.

Highly effective inhibition of Akabane virus replication by siRNA genes.

Since 2002 there has been a rise in arthrogryposis/hydranencephaly (AGH) incidence in Israel, caused by Akabane (AKA) and, possibly, Aino viruses. To test the ability to control the disease, three siRNA genes targeted to the S genome segment were designed and prepared in the form of siRNA cassettes. For the design all published S segment were aligned and two conserved target sequences with 100% homology were chosen. A third conserved target that was found exhibited only one base change found in the two Australian isolates and was also designed and tested. It was demonstrated that cells transfected with single siRNA genes showed 99% inhibition, as measured by real-time RT-PCR, virus titration and immunofluorescence. When cells were transfected with all three genes together the inhibition levels were increased and reached almost 100%.

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.

Bovine necrotic vulvovaginitis associated with Porphyromonas levii.

An outbreak of bovine necrotic vulvovaginitis associated with Porphyromonas levii, an emerging animal and human pathogen, affected 32 cows on a dairy farm in the northeast of Israel. Five animals had to be culled. This report appears to be the first that associates P. levii with bovine necrotic vulvovagnitis.