The Absence of Abdominal Pigmentation in Livestock Associated Culicoides following Artificial Blood Feeding and the Epidemiological Implication for Arbovirus Surveillance.

Culicoides midges (Diptera: Ceratopogonidae), the vectors of economically important arboviruses such as bluetongue virus and African horse sickness virus, are of global importance. In the absence of transovarial transmission, the parity rate of a Culicoides population provides imperative information regarding the risk of virus dispersal. Abdominal pigmentation, which develops after blood feeding and ovipositioning, is used as an indicator of parity in Culicoides. During oral susceptibility trials over the last three decades, a persistent proportion of blood engorged females did not develop pigment after incubation. The present study, combining a number of feeding trials and different artificial feeding methods, reports on this phenomenon, as observed in various South African and Italian Culicoides species and populations. The absence of pigmentation in artificial blood-fed females was found in at least 23 Culicoides species, including important vectors such as C. imicola, C. bolitinos, C. obsoletus, and C. scoticus. Viruses were repeatedly detected in these unpigmented females after incubation. Blood meal size seems to play a role and this phenomenon could be present in the field and requires consideration, especially regarding the detection of virus in apparent "nulliparous" females and the identification of overwintering mechanisms and seasonally free vector zones.

Vector Competence of Italian Populations of Culicoides for Some Bluetongue Virus Strains Responsible for Recent Northern African and European Outbreaks.

The distribution of Bluetongue virus (BTV) in Europe can be represented by two distinct and interconnected epidemiological systems (episystems), each characterized by different ecological characteristics and vector species. This study investigated the vector competence of Italian populations of Culicoides imicola and Culicoides obsoletus/scoticus to some representative BTV strains after artificial oral infection. The BTV strains were selected according to their ability to spread to one or both episystems and included BTV-4 ITA, responsible of the recent Italian and French BTV-4 outbreaks; the BTV-2 strain which caused the first BTV incursion in Italy, Corsica, and Balearic Islands; BTV-4 MOR, responsible for the epidemic in Morocco; and BTV-8, the strain which spread through Europe between 2006 and 2008. Blood-soaked cotton pledgets and Hemotek membrane feeder using Parafilm® membrane were used to artificially feed midges. For each population/strain, recovery rates (positive/tested heads) were evaluated using serogroup- and serotype-specific RT-PCR. The trial demonstrated that, except for the Abruzzo population of C. obsoletus/C. scoticus, which was refractory to BTV-4 MOR infection, all the investigated Culicoides populations are susceptible to the selected BTV strains and that, if prompt vaccination programs and restriction measures had not been implemented, BTV-2 and BTV-4 MOR could have spread all over Europe.

Mosquito species involved in the circulation of West Nile and Usutu viruses in Italy.

Usutu (USUV) and West Nile (WNV) are mosquito-borne Flavivirus emerged in Italy in 1996 and 1998, respectively, and reappeared 10 years later. The aim of this work is to review the Italian mosquito species found positive for WNV and USUV between 2008 and 2014. Moreover, the role of mosquitoes in promoting the overwintering of these viruses is discussed, as a result of the mosquito collections performed in Molise region between September 2010 and April 2011. Overall 99,000 mosquitoes were collected: 337 and 457 mosquito pools tested positive by real time reverse transcriptase polymerase chain reaction (real time RT-PCR) for WNV and USUV, respectively. West Nile virus was detected in pools of Culex pipiens s.l. (329), Ochlerotatus caspius (4), Culex modestus (2), and Culex spp. (2). Positive USUV pools were from Cx. pipiens s.l. (435), Aedes albopictus (12), Oc. caspius (5), Culex spp. (2), Anopheles maculipennis s.l. (1), Culiseta annulata (1), and Ochlerotatus detritus (1). In Molise region, 1,694 mosquitoes were collected, and USUV was identi ed in Cx. pipiens s.l., Cs. annulata, and Oc. detritus pools. This paper shows that Cx. pipiens s.l. is the mosquito species most involved in the WNV and USUV circulation in Italy, although other species would also support the spread of both the viruses during Winter.

Eco-climatic indicators for three Culicoides species of the Obsoletus complex in Italy.

Bluetongue, Schmallenberg and African horse sickness viruses are transmitted by vectors belonging to the genus Culicoides (Diptera: Ceratopogonidae). Among this genus, species of the Obsoletus complex play a crucial role in Europe. In Italy the complex includes three species: Culicoides obsoletus sensu strictu, Culicoides scoticus and Culicoides montanus. These three sibling species were identified in 80 collection sites across Italy through a multiplex PCR test. Sixty­four sites were classified with a predominant species through a Bayesian approach. Environmental, topographic and climatic factors together with landscape metrics were investigated in each species group using the non­parametric Mann­Whitney test. Culicoides obsoletus s.s. resulted the most widely distributed species across the peninsula (51 sites). This species was collected in many eco­climatic conditions, at a wide range of temperature and altitudes, mainly in anthropogenic landscape. Conversely C. scoticus collection sites were dominated by natural vegetated areas or forest, at medium higher altitudes, preferably in a wilder and more pristine environment (predominant species in 8 sites). Culicoides montanus was a rarer species, statistically predominant in only 3 sites located in Southern Italy, characterized by temperatures higher than the other species. These results, together with other on­going researches on the vector competence of the species belonging to the Obsoletus complex, and with investigation of larval and breeding habitats, could greatly improve epidemiological knowledge of possible spreading Culicoides­borne viruses.

The 'Culicoides obsoletus group' in Italy: relative abundance, geographic range, and role as vector for Bluetongue virus.

As Bluetongue virus (BTV) spread in Italy following its first incursion in 2000, it soon became apparent that, besides Culicoides imicola, additional species of the subgenus Avaritia were involved as vectors, namely one or more of the species that belong to the so-called 'Culicoides obsoletus group', which comprises C. dewulfi, C. chiopterus, C. obsoletus sensu stricto, C. scoticus and C. montanus; the three last named species are considered generally as forming the Obsoletus complex. This study presents the findings made over the last decade and more, within the Italian entomological surveillance program for Bluetongue. It describes the integrated morphological and molecular approach used to identify the species of the 'C. obsoletus group', maps in detail their relative abundances and geographic ranges in Italy, clarifies the hitherto unknown comparative seasonal abundances of C. obsoletus s.s. and C. scoticus in a site in Central Italy, and provides further details on the potential vector status of five species of the 'C. obsoletus group', with emphasis on C. obsoletus s.s., C. scoticus and C. montanus. Unlike the situation in Northern Europe, Culicoides dewulfi and C. chiopterus are uncommon to rare in Italy. In contrast, the Obsoletus complex occurs abundantly throughout Italy, with C. obsoletus s.s. being the most prevalent and ecologically adaptive of the three species making up the complex. A longitudinal study conducted at a site in Central Italy revealed that: (i) species of the Obsoletus complex prefer horses to sheep; (ii) their parity rates range from 10% (March) to 56% (November); (iii) throughout the year C. scoticus is consistently more abundant than C. obsoletus s.s.; (iv) abundances in both, C. obsoletus s.s. and C. scoticus, peak in May-June, with the peak of the latter species being more evident. Bluetongue virus was first isolated from wild caught midges of the Obsoletus complex in 2002. Thereafter, pools of selected parous midges collected across Italy, and during multiple outbreaks of BT, have been found consistently PCR-positive for the virus. More recently, viral RNA has been detected in field specimens of C. dewulfi, C. obsoletus s.s., C. scoticus and C. montanus.

Epizootic haemorrhagic disease in Italy: vector competence of indigenous Culicoides species and spatial multicriteria evaluation of vulnerability.

Epizootic haemorrhagic disease (EHD) is an infectious non-contagious viral disease transmitted by Culicoides, which affects wild and domestic ruminants. The disease has never been reported in Europe, however recently outbreaks of EHD occurred in the Mediterranean Basin. Consequently, the risk that Epizootic haemorrhagic disease virus (EHDV) might spread in Italy cannot be ignored. The aim of this study was to evaluate the risk of EHDV transmission in Italy, in case of introduction, through indigenous potential vectors. In Italy, the most spread and abundant Culicoides species associated to livestock are Culicoides imicola and the members of the Obsoletus complex. Culicoides imicola is a competent vector of EHDV, whereas the vector status of the Obsoletus complex has not been assessed yet. Thus, its oral susceptibility to EHDV was here preliminary evaluated. To evaluate the risk of EHDV transmission a geographical information system-based Multi-Criteria Evaluation approach was adopted. Distribution of vector species and host density were used as predictors of potential suitable areas for EHDV transmission, in case of introduction in Italy. This study demonstrates that the whole peninsula is suitable for the disease, given the distribution and abundance of hosts and the competence of possible indigenous vectors.

Vector species of Culicoides midges implicated in the 2012­2014 Bluetongue epidemics in Italy.

In 2012, serotypes 1 and 4 of bluetongue virus (BTV) entered and co­circulated in Sardinia. The following year, BTV­1 spread all over Sardinia and invaded Sicily and the Italian Tyrrenian coast. In 2014, this strain spread extensively in mainland Italy, causing severe outbreaks. In late 2014, BTV­4 was detected in Southern Italy (Apulia region). This study reports the detection of BTV in species of Culicoides (Diptera: Ceratopogonidae) collected in Italy during the epidemics between 2012 and 2014. A total of 2,925 pools (83,102 midges), sorted from 651 collections made on 339 affected farms of 12 Italian regions, were tested for the presence of BTV by real time polymerase chain reaction (RT­PCR). The study clearly shows that Culicoides imicola and Obsoletus complex have played a crucial role in the bluetongue (BT) epidemics in Italy in 2012­2014. Nevertheless, it also shows that other species may have played a role in transmitting BTV during these outbreaks. Culicoides dewulfi and at least 3 species of the Pulicaris complex, namely Culicoides pulicaris, Culicoides newsteadi and Culicoides punctatus, were found positive to BTV. Serotype 1 was detected in all species tested, whereas the BTV­4 was detected in the Obsoletus complex, C. imicola, and C. newsteadi.

Orbivirus detection from Culicoides collected on African horse sickness outbreaks in Namibia.

African horse sickness (AHS), a non-contagious infectious disease caused by a RNA virus in the Orbivirus genus within the Reoviridae family affecting all equids, is endemic in sub-Saharan Africa. The virus is transmitted by some species of biting midges in the genus Culicoides (Diptera: Ceratopogonidae). In April 2011, 8 Culicoides collections were performed in 6 districts of 4 regions of the Republic of Namibia (Africa), all within a 400 km radious from the capital Windhoek. Six farms - Khomas (Windhoek and Steinhausen), Erongo (Karibib and Omaruru), Otjozondjupa (Okahandja), and Omaheke (Gobabis) involved in the AHS outbreaks, were sampled. Overall 194,211 Culicoides were collected and identified. Culicoides imicola was largely the most abundant species at all farms (99.4%). A total of 18,687 parous and gravid Culicoides females were assayed for AHS virus (AHSV) by real time RT-PCR. Of the 248 assayed pools, 227 consisted of C. imicola, 13 of Culicoides pycnostictus and 5 of Schultzei complex. Only 1 pool each of Culicoides nivosus, Culicoides leucostictus, and Culicoides tropicalis was assayed. Of the 248 pools examined by real time RT-PCR, 81 tested positive for AHSV, all consisting of C. imicola collected at Omaruru, resulting in a field vector infection rate of 0.91%. No viable AHSV could be isolated from 88 of the tested pools (n = 1,463). However, bluetongue virus (BTV) serotype-1 and 10 were isolated from 3 of these pools, each consisting of 100 C. imicola collected at Windhoek. The present study confirms the relative low infection prevalence in field collected Culicoides and the strict relationship between the high abundance of C. imicola and outbreaks of AHSV.

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.

European surveillance for West Nile virus in mosquito populations.

A wide range of arthropod-borne viruses threaten both human and animal health either through their presence in Europe or through risk of introduction. Prominent among these is West Nile virus (WNV), primarily an avian virus, which has caused multiple outbreaks associated with human and equine mortality. Endemic outbreaks of West Nile fever have been reported in Italy, Greece, France, Romania, Hungary, Russia and Spain, with further spread expected. Most outbreaks in Western Europe have been due to infection with WNV Lineage 1. In Eastern Europe WNV Lineage 2 has been responsible for human and bird mortality, particularly in Greece, which has experienced extensive outbreaks over three consecutive years. Italy has experienced co-circulation with both virus lineages. The ability to manage this threat in a cost-effective way is dependent on early detection. Targeted surveillance for pathogens within mosquito populations offers the ability to detect viruses prior to their emergence in livestock, equine species or human populations. In addition, it can establish a baseline of mosquito-borne virus activity and allow monitoring of change to this over time. Early detection offers the opportunity to raise disease awareness, initiate vector control and preventative vaccination, now available for horses, and encourage personal protection against mosquito bites. This would have major benefits through financial savings and reduction in equid morbidity/mortality. However, effective surveillance that predicts virus outbreaks is challenged by a range of factors including limited resources, variation in mosquito capture rates (too few or too many), difficulties in mosquito identification, often reliant on specialist entomologists, and the sensitive, rapid detection of viruses in mosquito pools. Surveillance for WNV and other arboviruses within mosquito populations varies between European countries in the extent and focus of the surveillance. This study reviews the current status of WNV in mosquito populations across Europe and how this is informing our understanding of virus epidemiology. Key findings such as detection of virus, presence of vector species and invasive mosquito species are summarized, and some of the difficulties encountered when applying a cost-effective surveillance programme are highlighted.

Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM.

We identified a key oncogenic pathway underlying neuroblastoma progression: specifically, MYCN, expressed at elevated level, transactivates the miRNA 17-5p-92 cluster, which inhibits p21 and BIM translation by interaction with their mRNA 3' UTRs. Overexpression of miRNA 17-5p-92 cluster in MYCN-not-amplified neuroblastoma cells strongly augments their in vitro and in vivo tumorigenesis. In vitro or in vivo treatment with antagomir-17-5p abolishes the growth of MYCN-amplified and therapy-resistant neuroblastoma through p21 and BIM upmodulation, leading to cell cycling blockade and activation of apoptosis, respectively. In primary neuroblastoma, the majority of cases show a rise of miR-17-5p level leading to p21 downmodulation, which is particularly severe in patients with MYCN amplification and poor prognosis. Altogether, our studies demonstrate for the first time that antagomir treatment can abolish tumor growth in vivo, specifically in therapy-resistant neuroblastoma.