Detection of African Swine Fever Virus Antibodies in Serum and Oral Fluid Specimens Using a Recombinant Protein 30 (p30) Dual Matrix Indirect ELISA

In the absence of effective vaccine(s), control of African swine fever caused by African swine fever virus (ASFV) must be based on early, efficient, cost-effective detection and strict control and elimination strategies. For this purpose, we developed an indirect ELISA capable of detecting ASFV antibodies in either serum or oral fluid specimens. The recombinant protein used in the ELISA was selected by comparing the early serum antibody response of ASFV-infected pigs (NHV-p68 isolate) to three major recombinant polypeptides (p30, p54, p72) using a multiplex fluorescent microbead-based immunoassay (FMIA). Non-hazardous (non-infectious) antibody-positive serum for use as plate positive controls and for the calculation of sample-to-positive (S:P) ratios was produced by inoculating pigs with a replicon particle (RP) vaccine expressing the ASFV p30 gene. The optimized ELISA detected anti-p30 antibodies in serum and/or oral fluid samples from pigs inoculated with ASFV under experimental conditions beginning 8 to 12 days post inoculation. Tests on serum (n = 200) and oral fluid (n = 200) field samples from an ASFV-free population demonstrated that the assay was highly diagnostically specific. The convenience and diagnostic utility of oral fluid sampling combined with the flexibility to test either serum or oral fluid on the same platform suggests that this assay will be highly useful under the conditions for which OIE recommends ASFV antibody surveillance, i.e., in ASFV-endemic areas and for the detection of infections with ASFV isolates of low virulence.

The impact and control of malignant catarrhal fever in Tanzania

Malignant Catarrhal Fever (MCF), an often-lethal infectious disease, presents as a variable complex of lesions in susceptible ungulate species. The disease is caused by a -herpesvirus following transmission from an inapparent carrier host. Two major epidemiological forms exist: wildebeest-associated MCF (WA-MCF), in which the virus is transmitted to susceptible species by wildebeest calves less than approximately four months of age, and sheepassociated MCF (SA-MCF) in which the virus is spread by sheep (primarily adolescents). Due to the lack of an in-vitro propagation system for the causative agent of the more economically significant SA-MCF, and with the expectation that cross-protective immunity may be provided, vaccine development has focused on the more easily propagated alcelaphine herpesvirus-1 (AlHV-1) that causes WA-MCF. In 2008 a direct viral challenge trial showed that a novel vaccine, employing an attenuated AlHV-1 (atAlHV-1) `C5000 virus strain, protected British Friesian-Holstein (FH) cattle against an intranasal challenge with virulent AlHV-1 `C5000 virus. For cattle keeping people living near wildebeest calving areas in sub-Saharan Africa an effective vaccine would have value as it would release them from the costly annual disease avoidance strategy of having to move their herds away from the oncoming wildebeest. On the other hand, an effective vaccine will release herd owners from the need to avoid MCF, allowing them to graze their cattle alongside wildebeest on the highly nutritious pastures of the calving areas. As such conservationists have raised concerns that the development of a vaccine might lead to detrimental grazing competition. The principle objective of this study was to test the novel vaccine on Tanzanian shorthorn zebu cross cattle (SZC).We did this firstly using a natural challenge field trial (Chapter Two) which demonstrated that immunisation with the atAlHV-1 vaccine was well tolerated and induced an oro-nasopharyngeal AlHV-1-specific and -neutralising antibody response. This resulted in an immunity in SZC cattle that was partially protective and reduced naturally transmitted infection by 56%. We also demonstrated that non-fatal infections occurred with a much higher frequency than previously thought. Because the calculated efficacy of the vaccine was less than that seen in British FH cattle we wanted to determine whether host factors, particular to SZC cattle, had impacted the outcomes of the field trial. To do this we repeated the 2008 direct viral challenge trial using SZC cattle (Chapter Four). During this trial we also investigated whether the recombinant bacterial flagellin monomer (FliC), when used as an adjuvant, might improve the vaccine’s efficacy. The findings from this trial indicated that direct challenge with pathogenic AlHV-1 is effective at inducing MCF in SZC cattle and that FliC is not an appropriate adjuvant for this vaccine. Furthermore, with less control group cattle dying of MCF than expected we speculate that SZC cattle may have a degree of resistance to MCF that affords them protection from infection and developing fatal disease. In Chapter Three we investigated aspects of the epidemiology of MCF, specifically whether wildebeest placenta, long implicated by Maasai cattle owners as a source of MCF, might play a role in viral transmission. Additionally, through comparative sequence analysis, at two specific genes (A9.5 and ORF50) of wild-type and atAlHV-1, we investigated whether the `C5000 strain, the source of which was taken from Africa more than 40 years ago, was appropriate for vaccine development. The detection of AlHV-1 virus in approximately 50% of placentae indicated that infection can occur in-utero and that this tissue might play a role in disease transmission. And, despite describing three new alleles of the A9.5 gene (supporting previous evidence that this gene is polymorphic and encodes a secretory protein with interleukin-4 as the major homologue), the observation that the most frequently detected haplotypes, in both wild-type and attenuated AlHV-1, were identical suggests that AlHV-1 has a slow molecular clock and that the attenuated strain was appropriate for vaccine development. In Chapter Five we present the first quantitative assessment of the annual MCF avoidance costs that Maasai pastoralists incur. In particular we estimated that as a result of MCF avoidance 64% of the total daily milk yield during the MCF season was not available to be used by the 81% of the family unit remaining at the permanent boma. This represents an upper-bound loss of approximately 8% of a household0s annual income. Despite these considerable losses we concluded that, given an incidence of fatal MCF in cattle living in wildebeest calving areas of 5% to 10%, if herd owners were to stop trying to avoid MCF by allowing their cattle to graze alongside wildebeest, any gains made through increased availability of milk, improved body condition and reduced energy demands would be offset by an increase in MCF-incidence. With the development of an effective vaccine, however, this alternative strategy might become optimal. The overall conclusion we draw therefore is that, despite the substantial costs incurred each year avoiding MCF, the partial protection afforded by the novel vaccine strategy is not sufficient to warrant a wholesale change in disease avoidance strategy. Nonetheless, even the partial protection provided by this vaccine could be of value to protect animals that cannot be moved, for example where some of the herd remain at the boma to provide milk or where land-use changes make traditional disease avoidance difficult. Furthermore, the vaccine may offer a feasible solution to some of the current land-use challenges and conflicts, providing a degree of protection to valuable livestock where avoidance strategies are not possible, but with less risk of precipitating the potentially damaging environmental consequences, such as overgrazing of highly nutritious seasonal pastures, that might result if herd owners decide they no longer need to avoid wildebeest.

Proton-transfer versus nontransfer in compounds of the diazo-dye precursor 4-(phenyldiazenyl) aniline (aniline yellow) with strong organic acids: the 5-sulfosalicylate and the dichroic benzenesulfonate salts, and the 1:2 adduct with 3,5-dinitrobenzoic

The structures of two 1:1 proton-transfer red-black dye compounds formed by reaction of aniline yellow [4-(phenyldiazenyl)aniline] with 5-sulfosalicylic acid and benzenesulfonic acid, and a 1:2 nontransfer adduct compound with 3,5-dinitrobenzoic acid have been determined at either 130 or 200 K. The compounds are 2-(4-aminophenyl)-1-phenylhydrazin-1-ium 3-carboxy-4-hydroxybenzenesulfonate methanol solvate, C12H12N3+.C7H5O6S-.CH3OH (I), 2-(4-aminophenyl)-1-hydrazin-1-ium 4-(phenydiazinyl)anilinium bis(benzenesulfonate), 2C12H12N3+.2C6H5O3S-, (II) and 4-(phenyldiazenyl)aniline-3,5-dinitrobenzoic acid (1/2) C12H11N3.2C~7~H~4~N~2~O~6~, (III). In compound (I) the diaxenyl rather than the aniline group of aniline yellow is protonated and this group subsequently akes part in a primary hydrogen-bonding interaction with a sulfonate O-atom acceptor, producing overall a three-dimensional framework structure. A feature of the hydrogen bonding in (I) is a peripheral edge-on cation-anion association involving aromatic C--H...O hydrogen bonds, giving a conjoint R1/2(6)R1/2(7)R2/1(4)motif. In the dichroic crystals of (II), one of the two aniline yellow species in the asymmetric unit is diazenyl-group protonated while in the other the aniline group is protonated. Both of these groups form hydrogen bonds with sulfonate O-atom acceptors and thee, together with other associations give a one-dimensional chain structure. In compound (III), rather than proton-transfer, there is a preferential formation of a classic R2/2(8) cyclic head-to-head hydrogen-bonded carboxylic acid homodimer between the two 3,5-dinitrobenzoic acid molecules, which in association with the aniline yellow molecule that is disordered across a crystallographic inversion centre, result in an overall two-dimensional ribbon structure. This work has shown the correlation between structure and observed colour in crystalline aniline yellow compounds, illustrated graphically in the dichroic benzenesulfonate compound.

Persistent chlamydial infections : role in inflammation and challenges for vaccine development.

The development of chlamydial vaccines continues to be a major challenge for researchers. While acute infections are the main target of vaccine development groups, Chlamydia is well known for its ability to establish chronic or persistent infections in its host. To date, little effort has focussed specifically on the challenges of vaccines to successfully combat the chronic or persistent phase of the disease and yet this will be a necessary aspect of any fully successful chlamydial vaccine. This short review specifically examines the phenomenon of chlamydial persistence and the unique challenges that this brings to vaccine development.

Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Phenyl-ring disorder in the two-dimensional hydrogen-bonded structure of the 1:1 proton-transfer salt of the diazo-dye precursor 4-(phenyldiazenyl)aniline (aniline yellow) with L-tartaric acid

The structure of the 1:1 proton-transfer compound from the reaction of L-tartaric acid with the azo-dye precursor aniline yellow [4-(phenylazo)aniline], 4-(phenyldiazenyl)anilinium hydrogen 2R,3R-tartrate C12H12N3+ . C4H6O6- has been determined at 200 K. The asymmetric unit of the compound contains two independent phenylazoanilinium cations and two hydrogen L-tartrate anions. The structure is unusual in that all four phenyl rings of both cations have identical 50% rotational disorder. The two hydrogen L-tartrate anions form independent but similar chains through head-to-tail carboxylic O--H...O~carboxyl~ hydrogen bonds [graph set C7] which are then extended into a two-dimensional hydrogen-bonded sheet structure through hydroxyl O--H...O hydrogen-bonding links. The anilinium groups of the phenyldiazenyl cations are incorporated into the sheets and also provide internal hydrogen-bonding extensions while their aromatic tails layer in the structure without significant interaction except for weak \p--\p interactions [minimum ring centroid separation, 3.844(3) \%A]. The hydrogen L-tartrate residues of both anions have the common short intramolecular hydroxyl O--H...O~carboxyl~ hydogen bonds. This work has provided a solution to the unusual disorder problem inherent in the structure of this salt as well as giving another example of the utility of the hydrogen tartrate in the generation of sheet substructures in molecular assembly processes.

Dengue fever and el nino-southern oscillation in Queensland, Australia : a time series predictive model

Background It remains unclear over whether it is possible to develop an epidemic forecasting model for transmission of dengue fever in Queensland, Australia. Objectives To examine the potential impact of El Niño/Southern Oscillation on the transmission of dengue fever in Queensland, Australia and explore the possibility of developing a forecast model of dengue fever. Methods Data on the Southern Oscillation Index (SOI), an indicator of El Niño/Southern Oscillation activity, were obtained from the Australian Bureau of Meteorology. Numbers of dengue fever cases notified and the numbers of postcode areas with dengue fever cases between January 1993 and December 2005 were obtained from the Queensland Health and relevant population data were obtained from the Australia Bureau of Statistics. A multivariate Seasonal Auto-regressive Integrated Moving Average model was developed and validated by dividing the data file into two datasets: the data from January 1993 to December 2003 were used to construct a model and those from January 2004 to December 2005 were used to validate it. Results A decrease in the average SOI (ie, warmer conditions) during the preceding 3–12 months was significantly associated with an increase in the monthly numbers of postcode areas with dengue fever cases (β=−0.038; p = 0.019). Predicted values from the Seasonal Auto-regressive Integrated Moving Average model were consistent with the observed values in the validation dataset (root-mean-square percentage error: 1.93%). Conclusions Climate variability is directly and/or indirectly associated with dengue transmission and the development of an SOI-based epidemic forecasting system is possible for dengue fever in Queensland, Australia.

The Certainty of Yellow

Novel excerpt and discussion, as presented at the creative writing panel at Ignite! 2010

Vectors and alternative hosts of tobacco yellow dwarf virus in southeastern Australia

Factors that determine the epidemiology of Tobacco yellow dwarf virus (TbYDV), including alternative host plants and insect vector(s), were assessed over three consecutive growing seasons at four field sites in Northeastern Victoria in commercial tobacco growing properties. In addition, these factors were assessed for one growing season at three bean growing properties. Overall, 23 leafhopper species were identified at the 7 sites, with Orosius orientalis as the predominant leafhopper. Of the leafhoppers collected, only O. orientalis and Anzygina zealandica tested positive for TbYDV by polymerase chain reaction (PCR). The population dynamics of O. orientalis was assessed using sweep net sampling over three growing seasons and a trimodal distribution was observed. Despite large numbers of O. orientalis occurring early in the growing season (September–October), TbYDV was only detected in these leafhoppers between late November and end of January. The peaks in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and were associated with warmer temperatures and lower rainfall. Spatial and temporal distribution of vegetation at selected sites was determined using quadrat sampling. Of the 40 plant species identified, TbYDV was detected only in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. The proportion of host and non-host availability for leafhoppers was associated with climatic conditions.

Characterisation of the tritrophic interactions between tobacco yellow dwarf virus, its vector and host-plants

Tobacco yellow dwarf virus (TbYDV, family Geminiviridae, genus Mastrevirus) is an economically important pathogen causing summer death and yellow dwarf disease in bean (Phaseolus vulgaris L.) and tobacco (Nicotiana tabacum L.), respectively. Prior to the commencement of this project, little was known about the epidemiology of TbYDV, its vector and host-plant range. As a result, disease control strategies have been restricted to regular poorly timed insecticide applications which are largely ineffective, environmentally hazardous and expensive. In an effort to address this problem, this PhD project was carried out in order to better understand the epidemiology of TbYDV, to identify its host-plant and vectors as well as to characterise the population dynamics and feeding physiology of the main insect vector and other possible vectors. The host-plants and possible leafhopper vectors of TbYDV were assessed over three consecutive growing seasons at seven field sites in the Ovens Valley, Northeastern Victoria, in commercial tobacco and bean growing properties. Leafhoppers and plants were collected and tested for the presence of TbYDV by PCR. Using sweep nets, twenty-three leafhopper species were identified at the seven sites with Orosius orientalis the predominant leafhopper. Of the 23 leafhopper species screened for TbYDV, only Orosius orientalis and Anzygina zealandica tested positive. Forty-two different plant species were also identified at the seven sites and tested. Of these, TbYDV was only detected in four dicotyledonous species, Amaranthus retroflexus, Phaseolus vulgaris, Nicotiana tabacum and Raphanus raphanistrum. Using a quadrat survey, the temporal distribution and diversity of vegetation at four of the field sites was monitored in order to assess the presence of, and changes in, potential host-plants for the leafhopper vector(s) and the virus. These surveys showed that plant composition and the climatic conditions at each site were the major influences on vector numbers, virus presence and the subsequent occurrence of tobacco yellow dwarf and bean summer death diseases. Forty-two plant species were identified from all sites and it was found that sites with the lowest incidence of disease had the highest proportion of monocotyledonous plants that are non hosts for both vector and the virus. In contrast, the sites with the highest disease incidence had more host-plant species for both vector and virus, and experienced higher temperatures and less rainfall. It is likely that these climatic conditions forced the leafhopper to move into the irrigated commercial tobacco and bean crop resulting in disease. In an attempt to understand leafhopper species diversity and abundance, in and around the field borders of commercially grown tobacco crops, leafhoppers were collected from four field sites using three different sampling techniques, namely pan trap, sticky trap and sweep net. Over 51000 leafhopper samples were collected, which comprised 57 species from 11 subfamilies and 19 tribes. Twentythree leafhopper species were recorded for the first time in Victoria in addition to several economically important pest species of crops other than tobacco and bean. The highest number and greatest diversity of leafhoppers were collected in yellow pan traps follow by sticky trap and sweep nets. Orosius orientalis was found to be the most abundant leafhopper collected from all sites with greatest numbers of this leafhopper also caught using the yellow pan trap. Using the three sampling methods mentioned above, the seasonal distribution and population dynamics of O. orientalis was studied at four field sites over three successive growing seasons. The population dynamics of the leafhopper was characterised by trimodal peaks of activity, occurring in the spring and summer months. Although O. orientalis was present in large numbers early in the growing season (September-October), TbYDV was only detected in these leafhoppers between late November and the end of January. The peak in the detection of TbYDV in O. orientalis correlated with the observation of disease symptoms in tobacco and bean and was also associated with warmer temperatures and lower rainfall. To understand the feeding requirements of Orosius orientalis and to enable screening of potential control agents, a chemically-defined artificial diet (designated PT-07) and feeding system was developed. This novel diet formulation allowed survival for O. orientalis for up to 46 days including complete development from first instar through to adulthood. The effect of three selected plant derived proteins, cowpea trypsin inhibitor (CpTi), Galanthus nivalis agglutinin (GNA) and wheat germ agglutinin (WGA), on leafhopper survival and development was assessed. Both GNA and WGA were shown to reduce leafhopper survival and development significantly when incorporated at a 0.1% (w/v) concentration. In contrast, CpTi at the same concentration did not exhibit significant antimetabolic properties. Based on these results, GNA and WGA are potentially useful antimetabolic agents for expression in genetically modified crops to improve the management of O. orientalis, TbYDV and the other pathogens it vectors. Finally, an electrical penetration graph (EPG) was used to study the feeding behaviour of O. orientalis to provide insights into TbYDV acquisition and transmission. Waveforms representing different feeding activity were acquired by EPG from adult O. orientalis feeding on two plant species, Phaseolus vulgaris and Nicotiana tabacum and a simple sucrose-based artificial diet. Five waveforms (designated O1-O5) were observed when O. orientalis fed on P. vulgaris, while only four (O1-O4) and three (O1-O3) waveforms were observed during feeding on N. tabacum and the artificial diet, respectively. The mean duration of each waveform and the waveform type differed markedly depending on the food source. This is the first detailed study on the tritrophic interactions between TbYDV, its leafhopper vector, O. orientalis, and host-plants. The results of this research have provided important fundamental information which can be used to develop more effective control strategies not only for O. orientalis, but also for TbYDV and other pathogens vectored by the leafhopper.

Situation Review of Barley Yellow Dwarf Virus in West Asia and North Africa

Symptoms of barley yellow dwarf (BYD) have been observed on cereals in nearly all countries of West Asia and North Africa. Its incidence. however, has varied during the last 15 years. Observations from field surveys are summarized. Since symptoms of barley yellow dwarf virus (BYDV) are of low diagnostic value, especially in wheat (Triticum aestivum L.), more precise qualitative and quantitative detection was derived by vector transmission and serology. In 1985 and 1986. preliminary surveys by enzyme-linked immunosorbent assay (ELlS A) indicated that BYDV incidence in the regions surveyed in Syria, Morocco, and Tunisia was around 7. 22. and 24%. respectively. By vector transmission PAV-, RPV-, and RMV-like isolates ofBYDV were identified in Morocco and the PAV-like isolate in Syria. By serology PAV-like isolates were identified in Ethiopia, Lebanon. Morocco. Syria. and Tunisia. and MA V-like isolates were identified from Morocco and Tunisia. The PAV-like type was the most common in all countries surveyed. Screening for BYDV resistance by natural infection has been carried out in a number of countries of the region during the last few years. Screening for resistance by aphid inoculation was initiated in Syria in 1986 at the International Center for Agricultural Research in the Dry Areas (ICARDA). Such screening is expected to follow in other countries of the region soon.

Survey of Spring Oats for Barley Yellow Dwarf Viruses in Illinois

During the spring of 1987, 1,215 samples of spring oats (Avena sativa L.) were collected in Madison, Champaign, Woodford, Warren, and DeKalb counties, Illinois. At each site on each of three sampling dates, 45 samples were collected (regardless of symptoms) in a W pattern in I ha and tested for the PAY, MAV, RPV, and RMV serotypes of barley yellow dwarf virus (BYDV) by direct doubleantibody sandwich enzyme-linked immunosorbent assay (ELISA). RMV was not detected at any location. PAY and RPV were detected at all locations, as early as 17 April in Champaign County. The incidences of P A V and RPV from all plants sampled ranged from 2 to 64% and from 2 to 88%, respectively. Highest incidences of both strains were in May samples [rom Woodford County. MAV was detected in lower incidences (2-16%) only in samples from the central region of the state (Champaign, Woodford, and Warren counties). The presence of MA V serotypes was confirmed in triple-antibody sandwich ELISA with the MA V -specific MAFF2 monoclonal antibody from L. Torrance. In the last previous survey for BYDV in Illinois during 1967-1968 (1), about 75% of the isolates were PAY and about 20% were RPV; single isolates of RMV and MAV were found. Twenty years later, 55% were PAY, 39% were RPV, and 6% were MAV.