Identification of diffusion routes of O/EA-3 topotype of foot-and-mouth disease virus in Africa and Western Asia between 1974 and 2019 - a phylogeographic analysis.

Foot-and-mouth disease (FMD) affects the livestock industry and socioeconomic sustainability of many African countries. The success of FMD control programs in Africa depends largely on understanding the dynamics of FMD virus (FMDV) spread. In light of the recent outbreaks of FMD that affected the North-Western African countries in 2018 and 2019, we investigated the evolutionary phylodynamics of the causative serotype O viral strains all belonging to the East-Africa 3 topotype (O/EA-3). We analyzed a total of 489 sequences encoding the FMDV VP1 genome region generated from samples collected from 25 African and Western Asian countries between 1974 and 2019. Using Bayesian evolutionary models on genomic and epidemiological data, we inferred the routes of introduction and migration of the FMDV O/EA-3 topotype at the inter-regional scale. We inferred a mean substitution rate of 6.64 × 10-3  nt/site/year and we predicted that the most recent common ancestor for our panel of samples circulated between February 1967 and November 1973 in Yemen, likely reflecting the epidemiological situation in under sampled cattle-exporting East African countries. Our study also reinforces the role previously described of Sudan and South Sudan as a frequent source of FMDVs spread. In particular, we identified two transboundary routes of O/EA-3 diffusion: the first from Sudan to North-East Africa, and from the latter into Israel and Palestine AT; a second from Sudan to Nigeria, Cameroon, and from there to further into West and North-West Africa. This study highlights the necessity to reinforce surveillance at an inter-regional scale in Africa and Western Asia, in particular along the identified migration routes for the implementation of efficient control measures in the fight against FMD.

Molecular Detection of Avian Influenza Virus in Wild Birds in Morocco, 2016-2019.

Avian influenza (AI) is a zoonotic disease significant to both public and animal health, caused by influenza virus A, and affects domestic poultry, wild birds, and mammals including humans. Aquatic birds are considered the natural reservoir of this virus. In 2016, Morocco experienced the first occurrence of low pathogenic H9N2 avian influenza virus (AIV) in poultry; however, no cases were reported in wild birds. The present study aimed to monitor the presence of AIV in wild birds in Morocco in order to trace the possible sources of the viruses affecting poultry. Between 2016 and 2019, 967 samples obtained from 480 birds representing 56 different wild bird species, 20 families, and 8 orders, mostly from Charadriiformes, Anseriformes, Pelecaniformes, and Passeriformes, were collected from various wetlands and relevant ornithologic sites in Morocco. These field samples consisted of 374 cloacal swabs, 321 tracheal swabs, 54 fecal samples, and 218 organ pools including the trachea, lung, liver, spleen, heart, intestine, and brain. The samples were examined for the presence of AIV using TaqMan-based real-time reverse transcriptase-PCR (rRT-PCR) targeting the matrix gene, followed by further subtyping rRT-PCR tests targeting the H1-H16 genes. The AI matrix gene was detected in 18 out of 967 samples (1.86%); positive samples were detected in 17 birds belonging to 10 bird species: two redshanks (Tringa totanus), one little stint (Calidris minuta), one ruddy turnstone (Arenaria interpres), one common snipe (Gallinago gallinago), one common greenshank (Tringa nebularia), one black-winged stilt (Himantopus himantopus), two black-headed gulls (Chroicocephalus ridibundus), one slender-billed gull (Chroicocephalus genei), six cattle egrets (Bubulcus ibis), and one Eurasian coot (Fulica atra). AIV was detected in 2 wetlands and 1 ornithologic site (Sidi Moussa Oualidia Complex, Smir lagoon and El Jadida Coast) and the highest positivity was revealed in fresh fecal samples (11.1%), indicating the suitability of this matrix for wild bird surveillance. Our results highlight that waders, gulls, and cattle egrets are the most affected species and may represent a potential risk for AI introduction in the poultry sector in Morocco. Regular monitoring of wild birds in Morocco, focusing in particular in the areas and species identified in this study as a high risk of virus circulation, should be implemented to anticipate and prevent possible AIV spread.

Detección molecular del virus de la influenza aviar en aves silvestres en Marruecos, entre los años 2016 al 2019. La influenza aviar (IA) es una enfermedad zoonótica importante para la salud pública y animal, causada por el virus de la influenza A y afecta a la avicultura comercial, las aves silvestres y los mamíferos, incluyendo a los humanos. Las aves acuáticas se consideran el reservorio natural de este virus. En el año 2016, Marruecos experimentó la primera aparición del virus de la influenza aviar de baja patogenicidad H9N2 en avicultura; sin embargo, no se notificaron casos en aves silvestres. El presente estudio tuvo como objetivo monitorear la presencia del virus de influenza aviar en aves silvestres en Marruecos con el fin de rastrear las posibles fuentes de los virus que afectan a las aves comerciales. Entre los años 2016 y 2019, se recolectaron 967 muestras de 480 aves que representan 56 especies diferentes de aves silvestres, 20 familias y 8 órdenes, principalmente de Charadriiformes, Anseriformes, Pelecaniformes y Passeriformes, de varios humedales y sitios ornitológicos relevantes en Marruecos. Estas muestras de campo consistieron en 374 hisopos cloacales, 321 hisopos traqueales, 54 muestras fecales y 218 conjuntos de órganos que incluyeron tráquea, pulmón, hígado, bazo, corazón, intestino y cerebro. Las muestras se examinaron para detectar la presencia del virus de la influenza aviar mediante transcripción reversa y PCR en tiempo real basada en TaqMan (rRT-PCR) que estivo dirigida al gene de la matriz, seguida de más pruebas de subtipificación de rRT-PCR dirigidas a los genes H1 al H16. El gene de la matriz de influenza aviar se detectó en 18 de 967 muestras (1.86%); Se detectaron muestras positivas en 17 aves pertenecientes a 10 especies de aves: dos archibebes comunes (Tringa totanus), un correlimos chico (Calidris minuta), un vuelvepiedras común (Arenaria interpres), una agachadiza común (Gallinago gallinago), un archibebe claro (Tringa nebularia), una cigüeñuela común (Himantopus himantopus), dos gaviotas reidoras (Chroicocephalus ridibundus), una gaviota picofina (Chroicocephalus genei), seis garzas ganaderas (Bubulcus ibis) y una focha común (Fulica atra). El virus de la influenza aviar se detectó en 3 de los 17 humedales (Complejo Sidi Moussa-Walidia, Costa El Jadida y Laguna Smir) y la mayor positividad se reveló en muestras fecales frescas (11.1%), lo que indica la idoneidad de esta matriz para la vigilancia de aves silvestres. Estos resultados destacan que las aves zancudas, las gaviotas y las garzas ganaderas son las especies más afectadas y pueden representar un riesgo potencial para la introducción de influenza aviar en el sector avícola de Marruecos. Se debe implementar un monitoreo regular de las aves silvestres en Marruecos, centrándose en particular en las áreas y especies identificadas en este estudio como de alto riesgo de circulación del virus, para anticipar y prevenir la posible propagación del virus de la influenza aviar.

Evaluation of West Nile Virus Diagnostic Capacities in Veterinary Laboratories of the Mediterranean and Black Sea Regions.

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities.

Monitoring Anti-NS1 Antibodies in West Nile Virus-Infected and Vaccinated Horses.

West Nile virus (WNV) is a zoonotic arboviral pathogen affecting humans, birds, and horses. Vaccines are available for veterinary use, which efficiently prevent the infection in horses. Most common diagnostic tools rely on the identification of the agent (RT-PCR, virus isolation), or on the detection of antibodies (IgM and IgG) recognizing structural proteins of the virus or neutralizing virus infection in cell cultures (virus-neutralization tests). The recent emergence of WNV in different parts of the world has resulted in an increase in the vaccination of horses in many countries. Methods for differentiation between infected and vaccinated animals ("DIVA" assays) would be useful for surveillance and control purposes but are still not available. A usual approach in this regard is the use of antibodies to nonstructural proteins as markers of nonvaccinated, infected animals, and the nonstructural NS1 protein of WNV has been proposed as a candidate for such a marker. The aim of this study was to test the hypothesis that NS1 can be a useful antigen in DIVA assays for differentiating WNV vaccinated and infected horses in field conditions. For that, we examined serum samples from either vaccinated and infected horses both from experimental infections/vaccinations (under controlled conditions) and from the field, exposed to natural infection or vaccinated in response to a risk of infection. The overall conclusion of the study is that NS1 antigen can effectively differentiate WNV infected from vaccinated horses in experimental (controlled) conditions, but this differentiation might be difficult depending on the conditions prevailing in the field.

Reconstructing the evolutionary history of pandemic foot-and-mouth disease viruses: the impact of recombination within the emerging O/ME-SA/Ind-2001 lineage.

Foot-and-mouth disease (FMD) is a highly contagious disease of livestock affecting animal production and trade throughout Asia and Africa. Understanding FMD virus (FMDV) global movements and evolution can help to reconstruct the disease spread between endemic regions and predict the risks of incursion into FMD-free countries. Global expansion of a single FMDV lineage is rare but can result in severe economic consequences. Using extensive sequence data we have reconstructed the global space-time transmission history of the O/ME-SA/Ind-2001 lineage (which normally circulates in the Indian sub-continent) providing evidence of at least 15 independent escapes during 2013-2017 that have led to outbreaks in North Africa, the Middle East, Southeast Asia, the Far East and the FMD-free islands of Mauritius. We demonstrated that sequence heterogeneity of this emerging FMDV lineage is accommodated within two co-evolving divergent sublineages and that recombination by exchange of capsid-coding sequences can impact upon the reconstructed evolutionary histories. Thus, we recommend that only sequences encoding the outer capsid proteins should be used for broad-scale phylogeographical reconstruction. These data emphasise the importance of the Indian subcontinent as a source of FMDV that can spread across large distances and illustrates the impact of FMDV genome recombination on FMDV molecular epidemiology.