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In 2018, Croatia reported the largest outbreak of West Nile virus (WNV) infections as well as the re-occurrence of human Usutu virus (USUV) infections. For the first time, fatal WNV and USUV infections were detected in wild birds. We analysed epidemiological characteristics and molecular epidemiology of WNV and USUV infections detected during 2018 transmission season. From April to November, 178 patients with neuroinvasive disease and 68 patients with febrile disease were tested for WNV and USUV. Viral RNA was detected in cerebrospinal fluid (CSF) and urine samples using a real-time RT- PCR. Positive samples were tested by nested RT- PCR and nucleotide sequencing. IgM/IgG antibodies were detected in serum/CSF samples using ELISA with confirmation of cross- reactive samples by virus neutralization test (VNT). WNV neuroinvasive disease was confirmed in 54 and WNV fever in seven patients from 10 continental Croatian counties. Areas affected in 2018 were those in which cases occurred in previous seasons, while in three areas human cases were reported for the first time. Phylogenetic analysis of six strains from patients residing in different geographic areas showed circulation of WNV lineage 2. In three patients, neuroinvasive USUV infection was confirmed by RT-PCR or VNT. Sequence analysis of one detected strain revealed USUV Europe 2 lineage. During the same period, a total of 2, 574 horse and 1, 069 poultry serum samples were tested for WNV antibodies using ELISA. Acute asymptomatic WNV infection (IgM antibodies) was documented in 20/0.7% horses. WNV IgG antibodies were found in 307/11.9% horses and in 125/12.7% poultry. WNV RNA was detected in two goshawks and USUV RNA was detected in one blackbird from north-western Croatia. In the Zagreb area, 3, 670 female mosquitoes were collected. One Culex pipiens pool collected in July tested positive for USUV RNA. Our results highlight the importance of continuous multidisciplinary 'One health' surveillance of these emerging arboviruses.

In this rapid communication, a novel atypical bluetongue virus (BTV) strain detected in goats in the Piedmont region (north-western Italy) is described. This strain, BTV-Z ITA2017, is most related in Seg-2/VP-2 (83.8% nt/82.7% aa) to strain TOV of BTV-25. Reactive antisera of goats positive by cELISA for BTV antibodies failed to neutralize a chimeric virus expressing the outermost protein of TOV. Infected animals displayed low levels of RNAemia and absence of clinical signs consistent with bluetongue infection, a scenario described in animals infected with atypical BTV strains.

Since 1998, southern Europe has experienced multiple incursions of different serotypes and topotypes of Bluetongue virus, a vector-borne transmitted virus, the causative agent of Bluetongue (BT), a major disease of ruminants. Some of these incursions originated from northern Africa, likely because of wind-blown dissemination of infected midges. In this report, we describe the detection and whole genome characterization of a novel BTV-3 strain identified in a symptomatic sheep in Tunisia. Sequences were immediately deposited with the GenBank Database under Accession Nos KY432369-KY432378. Alert and preparedness are requested to face the next vector seasons in northern Africa and the potential incursion of this novel strain in southern Europe.

Summary In the last fifteen years, West Nile Virus (WNV) has dramatically expanded its geographic range and is now considered the most widespread arbovirus in the world. In Tunisia, West Nile Fever (WNF) outbreaks were reported in humans in 1997, 2003 and 2012. Usutu Virus (USUV), which is a ‘new’ emerging Flavivirus antigenically close to WNV, has never been reported in Tunisia. A serological investigation in 284 equids was conducted in 2012 in the southern west region of the country to assess the presence and prevalence of the WNV and USUV infection. Of the 284 samples tested by competitive enzyme-linked immunoassay, 129 were positive. Of these, 120 (42.3%) had WNV-specific neutralizing antibodies. The prevalence was significantly higher in areas closer to the oasis compared with that of the surrounding arid areas. Antibody titres against USUV were also reported in 10 equids. This was the first evidence of USUV circulation in Tunisia. Data recorded by this study indicate that WNV and USUV have circulated/are circulating in the region and that there is an urgent need to adapt the current surveillance programmes to this new scenario.

Summary Since 2000, several incursions of bluetongue virus (BTV) occurred in the Mediterranean Basin involving European and surrounding Countries. The Middle East represents one of the most important gateways for the access of BTV in Europe. Limited data on the BTV situation in this area are available. In this perspective, an epidemiological survey on the presence of BTV in Lebanon was conducted. Of the 181 serum samples tested, 97 (mean = 53.6%; 95% CI: 46.3–60.7) resulted positive when tested for the presence of BTV antibodies by c-ELISA, of these 42 (mean = 42%; 95% CI: 32.8–51.8) serum samples were from sheep and 55 (mean = 67.9%; 95% CI: 57.1–77.1) serum samples were from goats. Fourteen blood samples (14/110; mean = 12.7%; 95% CI: 7.8–20.3), 6 (6/66; mean = 9.1%; 95% CI: 4.4–18.5) from sheep and 8 (8/44; mean = 18.2%; 95% CI: 9.6–32.0) from goats, were positive by qRT-PCR. The results with serum-neutralization assay and typing performed by RT-PCR confirmed that six BTV serotypes are currently circulating in Lebanon, and these serotypes are as follows: 1, 4, 6, 8, 16 and 24. This study is the first report that confirms the presence and circulation of BTV in Lebanon.

SummaryThe recent outbreak caused by Schmallenberg virus, which affected sheep, goatsand cattle in Europe, highlighted the importance of having a robust surveillanceplan capable of monitoring abortions and malformations in the livestock off-spring. In this context, bluetongue viruses (BTVs) represented and represent oneof the major threats to the European livestock industry. Aiming to improve theunderstanding on BTV cross placental transmission and serotype involvement, inthis retrospective study foetal spleens and/or brains of 663 ovines, 429 bovines,155 goats and 17 buffaloes were tested for the presence of BTV by virus isolation.BTV vaccine strains were isolated from 31 foetuses (2.4%; 95% CI: 1.7–3.4%): 24(3.6%; 95% CI: 2.4–5.3%) from ovine foetal tissues; 6 (1.4%; 95% CI: 0.6–3.0%)from bovine foetal tissues and 1 (0.6%; 95% CI: 0.2–3.5%) from the spleen of acaprine foetus. All foetuses were from animals vaccinated with either BTV-2 orBTV-2, and BTV-9 modified live vaccines (MLVs) produced by OnderstepoortBiological Products (OBP), South Africa. Among the 31 isolated vaccine strains,serotype 9 (n = 28) was more frequently isolated (P < 0.05) than serotype 2(n = 3). In two cases infectious vaccine strains were found in the foetal tissues2 months after the vaccine administration. Other pathogens known to be causa-tive agents of abortion in ruminants were not detected nor isolated. This studydemonstrates, for the first time, that BTV-2 and BTV-9 vaccine strains are able tocross the placental barrier of sheep, cattle and goats. BTV-2 and BTV-9 vaccinestrains are able to infect foetuses and cause abortions or malformations dependingon the period of pregnancy at the time of vaccination.IntroductionBluetongue (BT) is an infectious vector-borne disease ofruminants caused by bluetongue virus (BTV), an RNA-virus which belongs to the family Reoviridae, genusOrbivirus (Verwoerd and Erasmus, 2004). At present, BTVofficially includes 24 distinct serotypes (Verwoerd andErasmus, 2004) based upon the immunological propertiesof the VP2 protein. The putative 25th and 26th serotypeshave been proposed by Maan et al. (2011) and Chaignatet al. (2008). The disease is evident in sheep although clini-cal cases were reported in cattle (Toussaint et al., 2006).The recent European BTV-8 outbreaks demonstrated forthe first time the capability of the BTV-8 field strain tocross the placental barrier, causing the birth of viraemic off-spring (De Clercq et al., 2008). Before the BTV-8 outbreak,vertical transmission had been observed only for BTVvaccine or tissue culture-adapted strains. All the reportedBTV foetal infections had been associated to BTV-1, BTV-10, BTV-11 and BTV-17 vaccine strains (Johnson et al.,1992; Melville and Gard, 1992; MacLachlan et al., 2000;Kirkland and Hawkes, 2004). Since 2000, Italy has beenexperiencing the most severe outbreaks of BT everrecorded. The Italian government starting from May 2002

Summary Migratory birds are considered one of the main sources for West Nile virus (WNV) introduction into European countries. Following the WNV epidemic in the late summer of 1998 in a marshy area of Tuscany (Padule of Fucecchio), an extensive ornithological surveillance programme was carried out in the infected areas from 2006 to 2008. Several species of migratory and resident birds were trapped, sampled and serologically tested. The results of this surveillance programme gave a useful indication of potential sources of WNV re-introduction and spread into Italy. The area under study was also investigated and classified into ecological areas through satellite image processing. In August 2008, the WNV infection re-emerged in Italy in the area surrounding the Po river delta, involving three regions: Lombardy, Emilia Romagna and Veneto. Several surveillance activities were immediately put in place, including the extensive monitoring of wild birds found dead or trapped in the framework of other surveillance programmes. These activities were also prolonged in the 2009, when the virus circulation re-occurred at the border of the area already infected in 2008. The possible epidemiological role of the different species of migratory and resident birds is discussed, in relation to the different ecological patterns identified in the area and their potential ability to introduce, spread and support the endemization of WNV infection.

Summary To assess the immunogenicity of Zulvac ! 8 Bovis (a commercial inactivated vaccine against bluetongue virus serotype 8 ‐ BTV8) under field conditions, 71 cattle vaccinated according to manufacturer schedule in Verona province (Italy) were tested for the presence of BTV8 neutralizing antibodies at 21, 29, 36, 43, 49, 102 and 201 days post-vaccination (dpv). Another group of 528 BTV8 vaccinated cattle in Mantova province (Italy) was also tested once between 113 and 174 dpv. The vaccine was able to elicit an immune response in 69 (97.2%) and 346 (65.5%) animals of the Verona and Mantova groups, respectively.