Spatial and temporal dynamics of West Nile virus between Africa and Europe.

It is unclear whether West Nile virus (WNV) circulates between Africa and Europe, despite numerous studies supporting an African origin and high transmission in Europe. We integrated genomic data with geographic observations and phylogenetic and phylogeographic inferences to uncover the spatial and temporal viral dynamics of WNV between these two continents. We focused our analysis towards WNV lineages 1 (L1) and 2 (L2), the most spatially widespread and pathogenic WNV lineages. Our study shows a Northern-Western African origin of L1, with back-and-forth exchanges between West Africa and Southern-Western Europe; and a Southern African origin of L2, with one main introduction from South Africa to Europe, and no back introductions observed. We also noticed a potential overlap between L1 and L2 Eastern and Western phylogeography and two Afro-Palearctic bird migratory flyways. Future studies linking avian and mosquito species susceptibility, migratory connectivity patterns, and phylogeographic inference are suggested to elucidate the dynamics of emerging viruses.

West Nile Virus Lineage 2 Overwintering in Italy.

In January 2022, West Nile virus (WNV) lineage 2 (L2) was detected in an adult female goshawk rescued near Perugia in the region of Umbria (Italy). The animal showed neurological symptoms and died 15 days after its recovery in a wildlife rescue center. This was the second case of WNV infection recorded in birds in the Umbria region during the cold season, when mosquitoes, the main WNV vectors, are usually not active. According to the National Surveillance Plan, the Umbria region is included amongst the WNV low-risk areas. The necropsy evidenced generalized pallor of the mucous membranes, mild splenomegaly, and cerebral edema. WNV L2 was detected in the brain, heart, kidney, and spleen homogenate using specific RT-PCR. Subsequently, the extracted viral RNA was sequenced. A Bayesian phylogenetic analysis performed through a maximum-likelihood tree showed that the genome sequence clustered with the Italian strains within the European WNV strains among the central-southern European WNV L2 clade. These results, on the one hand, confirmed that the WNV L2 strains circulating in Italy are genetically stable and, on the other hand, evidenced a continuous WNV circulation in Italy throughout the year. In this report case, a bird-to-bird WNV transmission was suggested to support the virus overwintering. The potential transmission through the oral route in a predatory bird may explain the relatively rapid spread of WNV, as well as other flaviviruses characterized by similar transmission patterns. However, rodent-to-bird transmission or mosquito-to-bird transmission cannot be excluded, and further research is needed to better understand WNV transmission routes during the winter season in Italy.

Epidemiological and Evolutionary Analysis of West Nile Virus Lineage 2 in Italy.

West Nile virus (WNV) is a mosquito-borne virus potentially causing serious illness in humans and other animals. Since 2004, several studies have highlighted the progressive spread of WNV Lineage 2 (L2) in Europe, with Italy being one of the countries with the highest number of cases of West Nile disease reported. In this paper, we give an overview of the epidemiological and genetic features characterising the spread and evolution of WNV L2 in Italy, leveraging data obtained from national surveillance activities between 2011 and 2021, including 46 newly assembled genomes that were analysed under both phylogeographic and phylodynamic frameworks. In addition, to better understand the seasonal patterns of the virus, we used a machine learning model predicting areas at high-risk of WNV spread. Our results show a progressive increase in WNV L2 in Italy, clarifying the dynamics of interregional circulation, with no significant introductions from other countries in recent years. Moreover, the predicting model identified the presence of suitable conditions for the 2022 earlier and wider spread of WNV in Italy, underlining the importance of using quantitative models for early warning detection of WNV outbreaks. Taken together, these findings can be used as a reference to develop new strategies to mitigate the impact of the pathogen on human and other animal health in endemic areas and new regions.

West Nile Virus Lineage 1 in Italy: Newly Introduced or a Re-Occurrence of a Previously Circulating Strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008-2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory active.

Genetic Diversity of Rift Valley Fever Strains Circulating in Namibia in 2010 and 2011.

Outbreaks of Rift Valley fever (RVF) occurred in Namibia in 2010 and 2011. Complete genome characterization was obtained from virus isolates collected during disease outbreaks in southern Namibia in 2010 and from wildlife in Etosha National Park in 2011, close to the area where RVF outbreaks occurred in domestic livestock. The virus strains were sequenced using Sanger sequencing (Namibia_2010) or next generation sequencing (Namibia_2011). A sequence-independent, single-primer amplification (SISPA) protocol was used in combination with the Illumina Next 500 sequencer. Phylogenetic analysis of the sequences of the small (S), medium (M), and large (L) genome segments of RVF virus (RVFV) provided evidence that two distinct RVFV strains circulated in the country. The strain collected in Namibia in 2010 is genetically similar to RVFV strains circulating in South Africa in 2009 and 2010, confirming that the outbreaks reported in the southern part of Namibia in 2010 were caused by possible dissemination of the infection from South Africa. Isolates collected in 2011 were close to RVFV isolates from 2010 collected in humans in Sudan and which belong to the large lineage containing RVFV strains that caused an outbreak in 2006-2008 in eastern Africa. This investigation showed that the RVFV strains circulating in Namibia in 2010 and 2011 were from two different introductions and that RVFV has the ability to move across regions. This supports the need for risk-based surveillance and monitoring.

Complete Coding Sequences of Lumpy Skin Disease Virus Strains Isolated from Cutaneous Lesions in Namibian Cattle during 2016 Outbreaks.

Between September and October 2016, an outbreak of lumpy skin disease (LSD) was monitored in the Okakarara veterinary district of Namibia. The complete coding sequences were obtained for LSD virus isolates from skin nodules from two symptomatic animals.

Electrospun PLGA Fiber Diameter and Alignment of Tendon Biomimetic Fleece Potentiate Tenogenic Differentiation and Immunomodulatory Function of Amniotic Epithelial Stem Cells.

Injured tendons are challenging in their regeneration; thus, tissue engineering represents a promising solution. This research tests the hypothesis that the response of amniotic epithelial stem cells (AECs) can be modulated by fiber diameter size of tendon biomimetic fleeces. Particularly, the effect of electrospun poly(lactide-co-glycolide) (PLGA) fleeces with highly aligned microfibers possessing two different diameter sizes (1.27 and 2.5 µm: ha1- and ha2-PLGA, respectively) was tested on the ability of AECs to differentiate towards the tenogenic lineage by analyzing tendon related markers (Collagen type I: COL1 protein and mRNA Scleraxis: SCX, Tenomodulin: TNMD and COL1 gene expressions) and to modulate their immunomodulatory properties by investigating the pro- (IL-6 and IL-12) and anti- (IL-4 and IL-10) inflammatory cytokines. It was observed that fiber alignment and not fiber size influenced cell morphology determining the morphological change of AECs from cuboidal to fusiform tenocyte-like shape. Instead, fleece mechanical properties, cell proliferation, tenogenic differentiation, and immunomodulation were regulated by changing the ha-PLGA microfiber diameter size. Specifically, higher DNA quantity and better penetration within the fleece were found on ha2-PLGA, while ha1-PLGA fleeces with small fiber diameter size had better mechanical features and were more effective on AECs trans-differentiation towards the tenogenic lineage by significantly translating more efficiently SCX into the downstream effector TNMD. Moreover, the fiber diameter of 1.27 µm induced higher expression of pro-regenerative, anti-inflammatory interleukins mRNA expression (IL-4 and IL-10) with favorable IL-12/IL-10 ratio with respect to the fiber diameter of 2.5 µm. The obtained results demonstrate that fiber diameter is a key factor to be considered when designing tendon biomimetic fleece for tissue repair and provide new insights into the importance of controlling matrix parameters in enhancing cell differentiation and immunomodulation either for the cells functionalized within or for the transplanted host tissue.

Detection of enzootic circulation of a new strain of West Nile virus lineage 1 in sentinel chickens in the north of Tunisia.

Tunisia has experienced various West Nile disease outbreaks. Notwithstanding the serological and molecular confirmations in humans, horses and birds, the human surveillance system can still be improved. Three sentinel chicken flocks were placed in different Tunisian endemic regions and followed up from September 2016 to January 2017. A total of 422 sera from Sejnene (north of Tunisia), 392 from Moknine (east coast of Tunisia) and 386 from Tozeur (south of Tunisia) were tested for West Nile-specific antibodies and viral RNA. The WNV elisa positive rate in sentinel chickens in Sejnene was 10.7% (95% CI: 5.08-21.52). No positive samples were detected in Moknine. In Tozeur, the overall serological elisa positive rate during the study period was 9.8% (95% CI:4.35-21.03). West Nile virus nucleic acid was detected in two chickens in Sejnene.Phylogenetic analysis of one of the detected partial NS3 gene sequences showed that recent Tunisian WNV strain belong to WNV lineage 1 and is closely related to Italian strains detected in mosquitoes in 2016 and in a sparrow hawk in 2017. This report showed the circulation, first molecular detection and sequencing of WNV lineage 1 in chickens in the north of Tunisia and highlights the use of poultry as a surveillance tool to detect WNV transmission in a peri-domestic area.

Assessing the role of migratory birds in the introduction of ticks and tick-borne pathogens from African countries: An Italian experience.

The continuous flow of billions of birds between Africa and Europe creates an "ecological bridge" between physically remote areas. Migratory birds fly south from their breeding grounds during late summer/fall and fly back in spring. These movements regulate the spread of internal and external parasites, as well as pathogens of potential public health concern. The aim of the present study was to investigate the possible introduction of exotic tick species and tick-borne pathogens into Europe via migratory birds. At the bird observatory of Ventotene island (Italy), 443 feeding ticks were collected from 249 birds captured and ringed during their northbound migration in spring 2013. Each tick was identified by morphological and molecular methods and then tested for bacterial and viral pathogens: Borrelia burgdorferi s.l., Rickettsia spp., Ehrlichia ruminantium and Coxiella burnetii, Crimean Congo haemorrhagic fever virus (CCHFV) and Flavivirus. Morphological and molecular identification confirmed Hyalomma rufipes as the most abundant species among the collected arthropods (366/443; 82.6%) followed by Hyalomma marginatum (10/433; 2.3%). Rickettsia aeschlimannii was identified in 158 ticks, while one engorged Amblyomma variegatum nymph was infected with Rickettsia africae. The other bacteria were not detected in any specimen. Among viruses, RNA belonging to West Nile virus and other Flavivirus were detected whereas all ticks were negative for CCHFV RNA. These results confirm how migratory birds play a role in carrying Rickettsia-infected ticks, as well as viruses of zoonotic importance, from Africa into Europe. To what extent tick species are capable of establishing a permanent population once introduced in naïve areas, is far from defined and deserve further investigation.

Crimean-Congo Hemorrhagic Fever Virus Genome in Tick from Migratory Bird, Italy.

We detected Crimean-Congo hemorrhagic fever virus in a Hyalomma rufipes nymph collected from a whinchat (Saxicola rubetra) on the island of Ventotene in April 2017. Partial genome sequences suggest the virus originated in Africa. Detection of the genome of this virus in Italy confirms its potential dispersion through migratory birds.

A Web Geographic Information System to share data and explorative analysis tools: The application to West Nile disease in the Mediterranean basin.

BACKGROUND: In the last decades an increasing number of West Nile Disease cases was observed in equines and humans in the Mediterranean basin and surveillance systems are set up in numerous countries to manage and control the disease. The collection, storage and distribution of information on the spread of the disease becomes important for a shared intervention and control strategy. To this end, a Web Geographic Information System has been developed and disease data, climatic and environmental remote sensed data, full genome sequences of selected isolated strains are made available. This paper describes the Disease Monitoring Dashboard (DMD) web system application, the tools available for the preliminary analysis on climatic and environmental factors and the other interactive tools for epidemiological analysis. METHODS: WNV occurrence data are collected from multiple official and unofficial sources. Whole genome sequences and metadata of WNV strains are retrieved from public databases or generated in the framework of the Italian surveillance activities. Climatic and environmental data are provided by NASA website. The Geographical Information System is composed by Oracle 10g Database and ESRI ArcGIS Server 10.03; the web mapping client application is developed with the ArcGIS API for Javascript and Phylocanvas library to facilitate and optimize the mash-up approach. ESRI ArcSDE 10.1 has been used to store spatial data. RESULTS: The DMD application is accessible through a generic web browser at https://netmed.izs.it/networkMediterraneo/. The system collects data through on-line forms and automated procedures and visualizes data as interactive graphs, maps and tables. The spatial and temporal dynamic visualization of disease events is managed by a time slider that returns results on both map and epidemiological curve. Climatic and environmental data can be associated to cases through python procedures and downloaded as Excel files. CONCLUSIONS: The system compiles multiple datasets through user-friendly web tools; it integrates entomological, veterinary and human surveillance, molecular information on pathogens and environmental and climatic data. The principal result of the DMD development is the transfer and dissemination of knowledge and technologies to develop strategies for integrated prevention and control measures of animal and human diseases.

Genetic characterization of Italian field strains of Schmallenberg virus based on N and NSs genes.

Following its first identification in Germany in 2011, the Schmallenberg virus (SBV) has rapidly spread to many other European countries. Despite the wide dissemination, the molecular characterization of the circulating strains is limited to German, Belgian, Dutch, and Swiss viruses. To fill this gap, partial genetic characterization of 15 Italian field strains was performed, based on S segment genes. Samples were collected in 2012 in two different regions where outbreaks occurred during distinct epidemic seasons. The comparative sequence analysis demonstrated a high molecular stability of the circulating viruses; nevertheless, we identified several variants of the N and NSs proteins not described in other SBV isolates circulating in Europe.

First evidence of West Nile virus lineage 2 circulation in Turkey.

In August 2014, a West Nile virus (WNV) strain belonging to lineage 2 was detected in the brain tissues of a 9 year old mare euthanised after showing severe clinical signs in Bursa region, Turkey. Phylogenetic analyses of 290 bp of NS3 coding region clustered the Turkish strain together with the 2010-2012 Greek isolates. Either IgG and IgM or IgG only WNV antibodies were detected in 2 and 11 horses, respectively, which were in the outbreak surrounding. No WNV RNA was detected in pools of 50 individuals of Culex pipiens (n = 2), Ochlerotatus caspius (n = 2), and Culex theileri (n = 1) collected in the infected premises.

Rift Valley Fever Virus among Wild Ruminants, Etosha National Park, Namibia, 2011.

After a May 2011 outbreak of Rift Valley fever among livestock northeast of Etosha National Park, Namibia, wild ruminants in the park were tested for the virus. Antibodies were detected in springbok, wildebeest, and black-faced impala, and viral RNA was detected in springbok. Seroprevalence was high, and immune response was long lasting.

First External Quality Assessment of Molecular and Serological Detection of Rift Valley Fever in the Western Mediterranean Region.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis which affects humans and a wide range of domestic and wild ruminants. The large spread of RVF in Africa and its potential to emerge beyond its geographic range requires the development of surveillance strategies to promptly detect the disease outbreaks in order to implement efficient control measures, which could prevent the widespread of the virus to humans. The Animal Health Mediterranean Network (REMESA) linking some Northern African countries as Algeria, Egypt, Libya, Mauritania, Morocco, Tunisia with Southern European ones as France, Italy, Portugal and Spain aims at improving the animal health in the Western Mediterranean Region since 2009. In this context, a first assessment of the diagnostic capacities of the laboratories involved in the RVF surveillance was performed. The first proficiency testing (external quality assessment--EQA) for the detection of the viral genome and antibodies of RVF virus (RVFV) was carried out from October 2013 to February 2014. Ten laboratories participated from 6 different countries (4 from North Africa and 2 from Europe). Six laboratories participated in the ring trial for both viral RNA and antibodies detection methods, while four laboratories participated exclusively in the antibodies detection ring trial. For the EQA targeting the viral RNA detection methods 5 out of 6 laboratories reported 100% of correct results. One laboratory misidentified 2 positive samples as negative and 3 positive samples as doubtful indicating a need for corrective actions. For the EQA targeting IgG and IgM antibodies methods 9 out of the 10 laboratories reported 100% of correct results, whilst one laboratory reported all correct results except one false-positive. These two ring trials provide evidence that most of the participating laboratories are capable to detect RVF antibodies and viral RNA thus recognizing RVF infection in affected ruminants with the diagnostic methods currently available.

The 2011 West Nile disease outbreak in Sardinia region, Italy.

In 2011, strains of West Nile Virus (WNV) belonging to lineage 1 spread for the first time in Sardinia region (Italy). In contrast to previous WNV Italian incursion, the strains were found in Culex modestus and, more surprisingly, they were able to cause severe clinical signs in the affected birds. Based on the partial sequence of the NS3 encoding gene, the Sardinian WNV strains demonstrated a high similarity with the other WNV strains recently detected in the Mediterranean Basin. Nonetheless, the 2011 Sardinian sequences were grouped in a distinct sub-cluster. Both the NS3-249P and NS3-249T genotypes were detected in the Sardinian outbreaks confirming that the co-circulation of different genotypes in the affected population might be common for WNV as for many RNA viruses. No association, however, was observed between virulence and viral genotype.

Rapid molecular detection and genotyping of West Nile Virus lineages 1 and 2 by real time PCR and melting curve analysis.

Following its spread in the USA, West Nile Virus (WNV) has reemerged in the Mediterranean basin with a renewed pathogenicity. The introduction of WNV lineage 2 in Europe and its co-circulation with lineage 1 has resulted in a continuously changing epidemiological scenario, highlighting the importance of differential detection of the two lineages. The paper describes a new real-time PCR method for the detection and genotyping of the two main lineages of WNV. The method requires a single pair of primers and probes and is based on the analysis of highly conserved consensus sequences detected in the 5' terminus of the viral genome.

The emergence of Schmallenberg virus across Culicoides communities and ecosystems in Europe.

Schmallenberg virus (SBV), a novel arboviral pathogen, has emerged and spread across Europe since 2011 inflicting congenital deformities in the offspring of infected adult ruminants. Several species of Culicoides biting midges (Diptera: Ceratopogonidae) have been implicated in the transmission of SBV through studies conducted in northern Europe. In this study Culicoides from SBV outbreak areas of mainland France and Italy (Sardinia) were screened for viral RNA. The role of both C. obsoletus and the Obsoletus complex (C. obsoletus and C. scoticus) in transmission of SBV were confirmed in France and SBV was also discovered in a pool of C. nubeculosus for the first time, implicating this species as a potential vector. While collections in Sardinia were dominated by C. imicola, only relatively small quantities of SBV RNA were detected in pools of this species and conclusive evidence of its potential role in transmission is required. In addition to these field-based studies, infection rates in colony-derived individuals of C. nubeculosus and field-collected C. scoticus are also examined in the laboratory. Rates of infection in C. nubeculosus were low, confirming previous studies, while preliminary examination of C. scoticus demonstrated that while this species can replicate SBV to a potentially transmissible level, further work is required to fully define comparative competence between species in the region. Finally, the oral competence for SBV of two abundant and widespread mosquito vector species in the laboratory is assessed. Neither Aedes albopictus nor Culex pipiens were demonstrated to replicate SBV to transmissible levels and appear unlikely to play a major role in transmission. Other vector competence data produced from studies across Europe to date is then comprehensively reviewed and compared with that generated previously for bluetongue virus.

First cases of Schmallenberg virus in Italy: surveillance strategies.

Following the first report of Schmallenberg virus (SBV) in the brain of a dystocic goat foetus in 2012 in Northern Italy, immediate response actions were adopted to avoid the virus circulation. The brain tested positive by 2 different one-step real-time RT-PCR protocols; these results were also confirmed by partial sequencing of the viral genome. At that time this was the first detection of the new Orthobunyavirus genus within the Bunyaviridae family in Italy. An epidemiological investigation in the involved farm was carried out in collaboration with the CESME - National Reference Centre for the study and verification of Foreign Animal Diseases (Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale', Italy). Epidemiological information on the flock was provided and analysed, whole blood and serum samples were also collected from all animals in the farm for both virological and serological investigations. All blood samples tested negative for SBV, whereas serological positive results were obtained by virus-neutralization (VN). Epidemiological investigations indicated the possible virus circulation in the area. The subsequent surveillance actions were mainly based on the standardization and reenforcement of passive surveillance protocols, a risk-based serological surveillance programme through VN and an entomological surveillance programme in the involved geographical areas were also put in place. Eventually SBV local circulation was confirmed by real time RT-PCR in 6 Culicoides pools, collected between September and November 2011 in 3 farms in the surroundings of the area of SBV outbreak.