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Context: West Nile virus (WNV) is an arthropod-borne virus considered a One Health challenge because of its increasing impact on human and animal health. It is one the most widely distributed viruses of the encephalitic Flaviviruses. It may cause severe neurological symptoms in humans and animals and is recognized as a serious public health problem also because of its impact on blood transfusion and organ transplantation. First identified in Africa in 1937, it was later introduced and spread in Italy, where in many regions it is now endemic, due to the increasingly favorable climatic and environmental conditions. Aim: The main objectives of this study, based on an interdisciplinary One Health approach, were: (1) to characterize the geographical distribution within specific host and vector populations in Africa; (2) to describe its phylogeographical patterns between Africa and Europe; (3) to define the genetic structure and epidemiology of Italian WNV strains, giving an insight of the viral circulation dynamics in the Italian territory. Methodology: Ecological and epidemiological studies were combined with molecular and phylogenetic analyses, carrying out field sampling activities, cellular culture, viral infection, immunofluorescent assay, multiplexed RT-PCR, sequencing, data analysis, and novel technique design. These activities were carried out both in Italy and in Senegal. Results: Our study evidences: (i) the circulation of several WNV lineages [Lineage 1 (L1), 2 (L2), 7 (L7), and 8 (L8)] in the African Continent; (ii) the presence of diverse competent mosquito vectors in Africa, mainly belonging to the Culex genus; (iii) the lack of vector competence studies for several other mosquito species found naturally infected with WNV in Africa; (iv) the need of more vector competence studies on ticks; (v) the circulation of WNV among humans, animals and vectors in at least 28 African countries; (vi) the lack of knowledge on the epidemiological situation of WNV for 19 African countries, and (vii) the importance of carrying out specific serological surveys in order to avoid possible bias on WNV circulation in Africa (objective 1). Furthermore, a new set of WNV L1 and L2 genome-specific primers for tiled-amplicon sequencing have been designed and a consistent dataset of 64 WNV L2 and 31 WNV L1 Italian genomes and of 3 WNV L2 and 7 WNV L1 Senegalese genome sequences from samples collected in Italy and Senegal between 2006 and 2022 has been produced. Twenty more WNV L1 and L2 Senegalese sequences obtained from samples collected in Senegal between 1985 and 2018 have been shared by the Institut Pasteur Dakar of Senegal and added to the dataset. This allowed the conduction of phylogenetic and phylogeographic analyses, evidencing: (viii) the presence of a strong viral connection between Africa and Europe, with intercontinental circulation supported by birds crossing international boundaries while migrating through the African-Eurasian flyways; (ix) the WNV L1 Western-Mediterranean cluster probable spread from Senegal, where the virus was first reported in 1979, to Italy, where the lineage first appeared in Europe in 1998, and to France in 2000, and the presence of back re-introductory events from Italy, Spain, and France to North and West Africa from the 2010s; and (x) the first African introduction of WNV L2 in Europe in Hungary in 2004, possibly from South African countries (objective 2). Our study also gives an insight of the dynamics of the viral circulation in Italy, demonstrating: (xi) the endemic presence of WNV L1 and L2 in part of Italy supported by resident wild birds and vector competent mosquitoes mainly belonging to the Culex genus; (xii) the current existence of two diverse WNV L1 strains circulating in Italy, one in the North-East, and one circulating intra-regionally in the Campania region; (xiii) suggested characteristic silent periods observed for WNV L1 in the country, with unnoticed circulation lasting sometimes for more than 10 years; (xiv) the 2022 WNV L1 increasing incidence of neurological disease cases in humans; (xv) the presence of genetically stable WNV L2 strains in Italy with continuous circulation throughout the time; (xvi) the presence of overwintering mechanisms supported by bird-to bird, rodent-to bird, or mosquito-to bird transmission routes; (xvii) the existence of WNV L1 and L2 co-infections in birds and mosquitoes; (xviii) the existence of a continuous transmission of the two strains between Western Mediterranean countries, supported by short distance migratory birds; and (xix) the crucial importance of the surveillance system other than the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases in Italy (objective 3). Conclusion: Our work points out the existence of high genetic diversity of WNV strains in Africa, the spread of L1 and L2 strains from Africa to Europe, and the existence of continuous transmission episodes among several Western-Mediterranean countries, with few recently suspected back introductory events from Europe to Africa. The progressive increase of the WNV L2 circulation both temporally and spatially in the Mediterranean countries and the WNV L1 re-appearance in Europe, both associated with a significant impact on humans and animal health, other than the strong WNV incidence in Italy and its endemization in part of its territory, evidence a solid WNV epidemic risk for Italy and a persistent threat for WNV spread into new areas. To predict and control future epidemics, it is crucial to constantly monitor the circulation and evolution of WNV in Europe and Africa, and to implement coordinated surveillance plans in both Continents, even in areas which are not currently affected.

See front file / Health Studies / DLITT ET PHIL (HEALTH ST)

A population survey was conducted from April through September 2002 on mosquito species occurring on the Ray Roberts Greenbelt, a riparian corridor used for public recreation on the Elm Fork of the Trinity River, in Denton County, Texas. ArcGIS software was used to set up a stratified random sampling design based on habitat parameters. Multivariate analyses of sampling data and climatic variables were used to describe spatial and temporal patterns of mosquito species. A total of 33 species were collected during this study belonging to the following genera: Aedes, Anopheles, Coquillettidia, Culex, Mansonia, Ochlerotatus, Orthopodomyia, Psorophora, Toxorhynchites, and Uranotaenia. Seasonal distributions of the dominant species revealed population fluctuations. Aedes vexans was the primary species collected in April and May, occurring in low numbers throughout the rest of the sampling period. Psorophora columbiae reached its highest population density in June, with a smaller peak occurring in late July. Present from May through the end of September, Culex erraticus was the most abundant species collected with major peaks in mid-June and the end of July. Abundance of Culex salinarius followed the same general trend as that for Cx. erraticus, but with smaller numbers. The specimens were tested for a variety of arboviruses by the Texas Department of Health. One pool of Cx. erraticus and Cx. salinarius, collected in August 2002, tested positive for West Nile virus. Variables that were important factors for determining dominant species abundance were temperature, wind speed, rain accumulation occurring one-week and two-weeks prior to sampling, number of day since last rain event, dew point, and average canopy coverage.

A number of emerging vector-borne diseases have the potential to be transmitted in the UK by native mosquitoes. Human infection by some of these diseases requires the presence of communities of multiple vector mosquito species. Mitigating the risk posed by these diseases requires an understanding of the spatial distributions of the UK mosquito fauna. Little empirical data is available from which to determine the distributions of mosquito species in the UK. Identifying areas at risk from mosquito-borne disease therefore requires statistical modelling to investigate and predict mosquito distributions. This thesis investigates the distributions of potential vector mosquitoes in the UK at landscape to national scales. A number of new methodological approaches for species distri- bution modelling are developed. These methods are then used to map and understand the distributions of mosquito communities with the potential to transmit diseases to humans. Chapter 2 reports the establishment of substantial populations of the West Nile virus (WNV) vector mosquito Culex modestus in wetlands in southern England. This represents a drastic shift in the species’ known range and an increase in the risk of WNV transmission where Cx. modestus is present. Chapter 3 develops and applies a new species interaction distribution model which identifies fish and ditch shrimp of the genus Palaemonetes as predators which may restrict the distribution of the potential WNV vector community in these wetlands. Chapter 4 develops a number of methods to make robust predictions of the probability of presence of a species from presence-only data, by eliciting and applying estimates of the species’ prevalence. Chapter 5 introduces a new Bayesian species distribution modelling approach which outperforms existing methods and has number of useful features for dealing with poor- quality data. Chapter 6 applies methods developed in the previous two chapters to produce the first high-resolution distribution maps of potential vector mosquitoes in the UK. These maps identify several wetland areas where vector communities exist which could maintain WNV transmission in birds and transmit it to humans. This thesis makes significant contributions to our understanding of the distributions of UK mosquito species. It also provides methods for species distribution modelling which could be widely applied in ecology and epidemiology.

O vírus do Oeste do Nilo (VON) é um Flavivírus capaz de infectar muitas espécies de vertebrados, incluindo o homem. Embora reconhecida desde 1940, esta virose nunca havia sido descrita nas Américas, onde emergiu nos Estados Unidos ao final da década de 1990, com numerosos casos de meningoencefalite em humanos. Posteriormente, sua transmissão por transfusão de sangue e órgãos foi comprovada, levando à implantação de testes moleculares (NAT) para a triagem de doadores nos EUA e Canadá a partir de 2003. Nos anos seguintes, o VON foi sendo progressivamente detectado em países como México, Panamá e áreas do Caribe, sugerindo sua iminente introdução na América do Sul. De fato, evidências sorológicas foram reveladas em cavalos e aves na Colômbia, Venezuela, Argentina e muito recentemente no pantanal mato-grossense (em cavalos). A vigilância epidemiológica para este agente é de grande importância para a saúde pública, visto o potencial de morbimortalidade deste vírus para humanos. Sendo assim este trabalho tem o objetivo de investigar a presença do RNA do VON em amostras de doadores de sangue, pacientes com meningoencefalite ou febre de origem indeterminada e soros e amostras cerebrais de equinos. Foram analisadas 2.202 doações de sangue do Amazonas (HEMOAM), 3.144 do Mato Grosso do Sul (HEMOSUL); líquido cefalorraquidiano de 51 pacientes com suspeita de meningoencefalite viral (Hospital das Clínicas/FMUSP, São Paulo) e soro de 198 pacientes com síndrome febril aguda, negativos para Dengue e Malária (Fundação de Medicina Tropical de Manaus). Além disto, 293 amostras de soros de equinos da região do Pantanal e 63 biópsias de tecido cerebral de cavalos que foram a óbito por encefalite de etiologia desconhecida. Estas amostras foram submetidas ao teste automatizado cobas TaqScreen WNV (Roche) na plataforma cobas s201 em sistema de pool de 6 unidades (doações de sangue) ou individualmente (pacientes). Todas as amostras apresentaram amplificação satisfatória do controle da reação, porém nenhuma apresentou resultado positivo para a presença do RNA do VON. Embora já exista evidência da exposição de equinos no Brasil ao VON, não parece haver até o momento, disseminação importante deste agente entre humanos e equinos, uma vez que o RNA viral não foi detectado nem em doadores de sangue e nem em equinos, incluindo os de cidades próximas aos locais onde cavalos soropositivos foram encontrados (Corumbá MS). / The West Nile Virus (WNV) is a Flavivirus able to infect many species of vertebrates, including man. Recognized since 1940, this virus had never been described in the Americas, which emerged in the United States at the end of the 1990s, with numerous cases of meningoencephalitis in humans. Later, transmission by transfusion of blood and organs was confirmed, leading to the deployment of molecular testing (NAT) for screening of donors in the U.S. and Canada since 2003. In the following years, WNV has been progressively detected in countries like Mexico, Panama and the Caribbean areas, suggesting their imminent introduction in South America In fact, serological evidence was revealed in horses and birds in Colombia, Venezuela and Argentina and most recently in Pantanal, Mato Grosso (horses). Epidemiological surveillance for this agent is of great importance to public health, given the potential morbidity and mortality of this virus to humans. Therefore this study aims to investigate the presence of WNV RNA in samples of blood donors, patients with meningoencephalitis or fever of unknown origin and serum and brain samples from horses. We analyzed 2202 blood donations from Amazon (HEMOAM), 3144 from Mato Grosso do Sul (HEMOSUL); cerebrospinal fluid of 51 patients with suspected viral encephalitis (Hospital das Clínicas / FMUSP, São Paulo) and serum samples from 198 patients with acute febrile syndrome, negative for Dengue and malaria (Foundation for Tropical Medicine in Manaus). In addition, more 293 serum samples from horses of the Pantanal and 63 biopsies of brain tissue from horses that died of encephalitis of unknown etiology. These samples were subjected to automated cobas TaqScreen WNV test (Roche) on the platform in cobas S201with a system of 6 units pool (blood donations) or individually (patients). All samples showed satisfactory control amplification, but none showed as positive for the presence of RNA VON. Although there is already evidence in horses in Brazil of exposure to WNV, there seems to be far that an important spread of this agent between humans and horses, since the viral RNA was not detected either in blood donors or in horses, including cities near the locations where seropositive horses were found (Corumbá - MS).

Le Virus du Nil Occidental (VNO) et le Virus de la Fièvre de la Vallée du Rift (VFVR) sont deux arbovirus transmis par le moustique Culex pipiens comprenant deux biotypes: pipiens et molestus. Au cours de ce projet, nous avons évalué la circulation du VNO au Liban dans des populations de moustiques, des humains, des chevaux et des poulets. Nous avons aussi évalué la compétence vectorielle des populations locales de Cx. pipiens à transmettre le VNO et le VFVR.Des moustiques ont été récoltés et testés pour la présence d’un gène spécifique du VNO. En plus, des sérums humains, de chevaux et de poulets ont été analysés pour rechercher des anticorps spécifiques par ELISA puis confirmés par neutralisation. En outre, des spécimens de Cx. pipiens ont été infectés avec la lignée 1 du VNO ou la souche de VFVR Clone 13. Ensuite, les taux d’infection, de dissémination et de transmission ont été déterminés à différents jours après infection des moustiques. La compétence vectorielle a été comparée entre les différents biotypes.Les résultats entomologiques ont révélé que Cx. pipiens est dominant (87.2%). Tous les moustiques analysés étaient négatifs pour le VNO. Les taux de séroprévalence étaient de 1.01% et 1.98% parmi les humains et les chevaux respectivement. De plus, Cx. pipiens s’est révélé bien plus compétent pour transmettre le VNO que le VFVR. Le biotype molestus est capable de transmettre le VNO plus tôt que celui de pipiens. Cette étude présente des preuves sur une faible circulation du VNO au Liban. Cx. pipiens s’est révélé compétent pour assurer cette transmission. Ainsi, il est essentiel d'établir des programmes de surveillance pour prévenir les éventuelles épidémies. / West Nile virus (WNV) and Rift Valley Fever virus (RVFV) are two emerging arboviruses that have never been reported in Lebanon. They can be transmitted by Culex pipiens mosquito species including two biotypes: pipiens and molestus. During this project, we assessed the circulation of WNV among mosquitoes, human, horse and chicken populations in Lebanon. Moreover, we evaluated, under experimental conditions, the capacity of local Cx. pipiens biotypes to transmit both viruses.Adult mosquitoes were collected, identified and tested to detect WNV RNA. Besides, human, horse and chicken blood samples were collected and screened for WNV antibodies using an in-house ELISA and then confirmed by neutralization assay. Moreover, local Cx. pipiens specimens were experimentally infected with WNV lineage 1 or RVFV Clone 13 strain. The viral infection, dissemination and transmission were then estimated at different days post infection.The vector competence was compared between Cx. pipiens biotypes.Entomological results revealed that 87.2% of collected adult mosquitoes were Cx. pipiens. Screened mosquitoes were negative for WNV. Seroprevalence rates were 1.01% and 1.98% among humans and horses respectively. Besides, local Cx. pipiens were highly competent for WNV transmission and to a lesser extent to RVFV. The molestus biotype was able to transmit WNV earlier than pipiens biotype.The present study provides new evidence of a low circulation of WNV among human and horses in Lebanon. Cx. pipiens is the suspected vector and is experimentally competent to ensure transmission. Therefore, there is a need to establish surveillance program to predict and prevent potential outbreaks.

The objectives of this study were to provide a comprehensive review of the risks and benefits of using adulticides to reduce risk of mosquito-borne disease (particularly West Nile Virus) transmission to humans, as well as to decrease annoyance from nuisance mosquitoes. The study was designed with two major research components, including: 1) an extensive literature review to determine the efficacy of adulticide use, the adverse effects of adulticide use, the impact of mosquitoes on community livability, and the risks and benefits of pesticide use in controlling mosquitoes; and 2) interviews with selected vector districts in seven states to determine effective and ineffective practices in mosquito management. This study has demonstrated that an integrated mosquito management program may be beneficial in reducing risk of disease transmission and mosquito annoyance when performed appropriately. The contribution of adulticiding to reducing mosquito-borne disease transmission, however, is unknown. Research is needed to: 1) further assess the ecological and human impacts of adulticides using the dose and exposure rates realistic to an adulticide program; 2) gain an understanding of the human and ecological impacts of aggregate and cumulative exposures to pesticides, especially for special populations, such as children; and 3) determine the contribution of adulticiding in interrupting or reducing the enzootic amplification of arboviruses, as well as the transmission of WNV to humans. / Graduation date: 2004

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.

West Nile Virus (WNV) is a mosquito-borne pathogen of global significance. It is active on several continents and is responsible for recent outbreaks of fever and fatal encephalitis in humans and horses. While highly virulent strains have been reported in Europe, North, Central and South America, only a benign subtype of WNV (Kunjin virus – KUNV) occurs in Australia. However, virulent, exotic WNV strains are seen as a significant threat to Australia due to the ease with which this virus can move between continents and the presence of suitable vectors and hosts already within Australia. KUNV and WNV subtypes are antigenically and genetically very closely related and cross-react in traditional serological tests. This cross-reactivity makes it very difficult to differentiate between KUNV and WNV infections using standard serological tests. The aim of this thesis was to identify immunogenic epitopes unique to KUNV or WNV and to use these epitopes in the development of a rapid assay that would enable the diagnosis of and surveillance for exotic virulent strains of WNV in Australia. The rapid diagnostic platform chosen was a red blood cell (RBC) agglutination assay that was originally patented and commercialised by AGEN Biomedical Ltd. The RBC agglutination assay reagent consists of the Fab region of a human erythrocyte-specific monoclonal antibody (mAb) conjugated to the epitope of interest (in this instance, a WNV-specific peptide). This bi-functional reagent causes the agglutination of the patient’s erythrocytes in the presence of WNV-specific antibody in the patient’s serum. Traditionally, these RBC agglutination reagents have been produced by chemical conjugation. However, a potentially easier and cheaper method involves the linking of the gene encoding the erythrocyte-specific antibody to that encoding the epitope to create a recombinant version of the bi-functional agglutination reagent through expression using prokaryotic or eukaryotic systems. To identify potential differential epitopes, 18 mAbs to WNV (NY99 strain) prM and envelope (E) proteins were assessed. One mAb (17D7) differentially recognised WNV and KUNV in ELISA and maintained recognition of its corresponding epitope upon reduction and carboxymethylation of the viral antigen, suggesting a continuous (linear) epitope. Using synthetic peptides, the epitope was mapped to a 19 amino acid sequence (WN19: E147-165) encompassing the WNV NY99 E protein glycosylation site at position 154. An amino acid substitution at position E156 of many KUNV strains abolishes this glycosylation moiety. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses that had been infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognised the recombinant peptide. In contrast, no reactivity with the recombinant peptide was observed by sera from horses infected with the unglycosylated WNV subtype, KUNV. Failure of most WNV- and MVEV-positive horse sera to recognise the epitope as a deglycosylated fusion protein (75% and 100% respectively) confirmed that the N-linked glycan is important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain. To assess the feasibility of using peptide WN19 in a rapid immunoassay, the peptide was recombinantly fused to a RBC (glycophorin)-specific single chain antibody (scFv) using previously published constructs which were developed for the bacterial expression of similar bi-functional reagents. To facilitate glycosylation of peptide WN19, the genes for the bi-functional agglutination reagents were subsequently cloned into eukaryotic expression vectors. An additional set of constructs were also produced in which the genes for the variable regions of the anti-RBC antibody were cloned into a vector for the secreted expression of an intact, humanised IgG1 molecule. Stable cell lines were produced for each of these constructs and secreted up to 700 ng/mL glycophorin-reactive antibody. The secreted recombinant protein could be harvested directly from the cell culture medium and used in RBC agglutination assays, where these bi-functional agglutination reagents could be cross-linked either with mAb 17D7 or by anti-peptide WN19 antibodies present in WNV-positive horse serum. The WNV NY99 prM protein was also identified as a useful marker of WNV-infection in horses, as well as a putative antigen to differentiate equine WNV NY99 and KUNV infections using Western blot. Two anti-WNV prM mAbs were also generated in this study and will be extremely valuable in future studies. Preliminary analysis of the prM epitope(s) bound by these mAbs and WNV-immune sera indicate that the binding site(s) is likely to be localised to pr and is conformational.