Domestic species, including equids, were introduced in the Galapagos Islands in the XIX century. Equine vector-borne diseases are circulating in South America but their occurrence in the Galapagos Island was unknown. The objective of this study was to detect the occurrence of West Nile virus (WNV), Usutu virus (USUV) and equine infectious anemia virus (EIAV) in the four Galapagos Islands raising equids if they were present at a prevalence >1%. Serum samples were collected from 411 equids belonging to 124 owners from April to July 2019. All the results were negative to the ELISA tests used suggesting that WNV, USUV and EIAV are not circulating in the equine population of the Galapagos Islands.

Arboviruses represent a public health concern in many European countries, including Italy, mostly because they can infect humans, causing potentially severe emergent or re-emergent diseases, with epidemic outbreaks and the introduction of endemic circulation of new species previously confined to tropical and sub-tropical regions. In this review, we summarize the Italian epidemiology of arboviral infection over the past 10 years, describing both endemic and imported arboviral infections, vector distribution, and the influence of climate change on vector ecology. Strengthening surveillance systems at a national and international level is highly recommended to be prepared to face potential threats due to arbovirus diffusion.

The Usutu virus (USUV), a neurotropic mosquito-borne flavivirus discovered in 1959 in South Africa, has spread over the last twenty years across the European continent. This virus follows an enzootic cycle involving mosquitoes and birds. This caused epizootics with significant bird mortality in Europe in 2016 and 2018. It can also occasionally infect humans and other mammals, including horses and bats, which act as incidental or dead-end hosts. The zoonotic risk associated with this succession of avian epizootics in Europe deserves attention, even if, to date, human cases remain exceptional. Human infection is most often asymptomatic or responsible for mild clinical symptoms. However, human Usutu infections have also been associated with neurological disorders, such as encephalitis and meningoencephalitis. One of the major complexities of the study of USUV pathogenesis is the presence of a great diversity of lineages which could co-circulate spatiotemporally. In this review we discuss several aspects of the circulation of Usutu virus in humans in Europe, the neurological disorders associated, involved viral lineages, and the issues and questions raised by their circulation.

The Usutu virus (USUV) has recently attracted the attention of scientists because of its rapid spread across Europe and its growth over the previous seasons in Italy. Here, we describe the first case of USUV infection in Asti, Piedmont region, Italy. The patient remained asymptomatic in the acute phase and during the early follow-up, despite a mild increase in liver enzymes. The prompt diagnosis in this patient was due to positive qualitative PCR for WNV blood-donor screening with negative RT-PCR of WNV and positive USUV-RNA following the confirmation test. Blood-donor screening and transmission risk monitoring are pivotal in following the spread of this Flavivirus in non-endemic countries, due to the high percentage of asymptomatic carriers.

West Nile virus (WNV) and Usutu virus (USUV) are mosquito-borne flaviviruses that have been associated with neurological diseases in humans and wild birds. Wild bird rescue centers are potential significant hot spots for avian infection surveillance, as recognized in the Italian Integrate National Surveillance Plan for Arboviruses. Here we report the results of a post-mortem active monitoring study conducted from November 2017 to October 2020 on animals hosted in five wild bird rescue centers of Central Italy. Five hundred seventy-six (n = 576) wild birds were tested by real-time polymerase chain reaction (RT-PCR) for the presence of WNV or USUV RNA fragments. No birds tested positive for USUV RNA (n = 0; 0.00%). Evidence of WNV RNA (Ct value = 34.36) was found in one bird (n = 1; 0.17%), an adult little grebe (Tachybaptus ruficollis subsp. ruficollis), that tested WNV positive in December 2019. This study highlights the strategic role of wildlife rescue centers in monitoring both the introduction and circulation of avian emerging zoonotic diseases. In addition, the presence of WNV during the cold season evidences the possible role of birds in overwintering mechanisms in the Italian territory and requires further investigations.

Cellular responses to stress generally lead to the activation of the endoplasmic reticulum-associated protein degradation (ERAD) pathway. Several lines of study support that ERAD may be playing a proviral role during flaviviral infection. A key host factor in ERAD is the valosin-containing protein (VCP), an ATPase which ushers ubiquitin-tagged proteins to degradation by the proteasome. VCP exhibits different proviral activities, such as engaging in the biogenesis of viral replication organelles and facilitating flavivirus genome uncoating after the viral particle entry. To investigate the possible antiviral value of drugs targeting VCP, we tested two inhibitors: eeyarestatin I (EEY) and xanthohumol (XAN). Both compounds were highly effective in suppressing Zika virus (ZIKV) and Usutu virus (USUV) replication during infection in cell culture. Further analysis revealed an unexpected virucidal activity for EEY, but not for XAN. Preincubation of ZIKV or USUV with EEY before inoculation to cells resulted in significant decreases in infectivity in a dose- and time-dependent manner. Viral genomes in samples previously treated with EEY were more sensitive to propidium monoazide, an intercalating agent, with 10- to 100-fold decreases observed in viral RNA levels, supporting that EEY affects viral particle integrity. Altogether, these results support that EEY is a strong virucide against two unrelated flaviviruses, encouraging further studies to investigate its potential use as a broad-acting drug or the development of improved derivatives in the treatment of flaviviral infection.

BackgroundDespite the known circulation of West Nile virus (WNV) and Usutu virus (USUV) in Slovakia, no formal entomological surveillance programme has been established there thus far.AimTo conduct contemporaneous surveillance of WNV and USUV in different areas of Slovakia and to assess the geographical spread of these viruses through mosquito vectors. The first autochthonous human WNV infection in the country is also described.MethodsMosquitoes were trapped in four Slovak territorial units in 2018 and 2019. Species were characterised morphologically and mosquito pools screened for WNV and USUV by real-time reverse-transcription PCRs. In pools with any of the two viruses detected, presence of pipiens complex group mosquitoes was verified using molecular approaches.ResultsAltogether, 421 pools containing in total 4,508 mosquitoes were screened. Three pools tested positive for WNV and 16 for USUV. USUV was more prevalent than WNV, with a broader spectrum of vectors and was detected over a longer period (June-October vs August for WNV). The main vectors of both viruses were Culex pipiens sensu lato. Importantly, WNV and USUV were identified in a highly urbanised area of Bratislava city, Slovakias\' capital city. Moreover, in early September 2019, a patient, who had been bitten by mosquitoes in south-western Slovakia and who had not travelled abroad, was laboratory-confirmed with WNV infection.ConclusionThe entomological survey results and case report increase current understanding of the WNV and USUV situation in Slovakia. They underline the importance of vector surveillance to assess public health risks posed by these viruses.

A serosurvey was carried out to assess emerging flavivirus exposure in zoo mammals in Spain and to determine the dynamics of seropositivity in species that were longitudinally sampled during the study period. Sera from 570 zoo animals belonging to 120 mammal species were collected at ten zoos (A-J) in Spain between 2002 and 2019. Twenty-one of these animals, belonging to ten different species, were sampled longitudinally at four of the zoos during the study period. Antigenically-related flavivirus antibodies were detected in 19 (3.3 %; 95 %CI: 2.0-5.2) of the 570 animals analyzed using bELISA. Seropositivity was observed in ten (8.3 %) of the 120 species tested. Five (23.8 %) of the 21 animals sampled more than once presented seropositivity in all samplings whereas seroconversion was only observed in one white rhinoceros (Ceratotherium simum). Flavivirus antibodies were found at six of the ten sampled zoos and in consecutive years between 2008 and 2018. Virus neutralization tests confirmed West Nile virus (WNV), Usutu virus (USUV) and tick-borne encephalitis virus (TBEV) infection in ten (1.8 %; 95 %CI: 0.7-2.8), five (0.9 %; 95 %CI: 0.1-1.6) and one (0.2 %; 95 %CI: 0.0-0.5) animal, respectively. Antibodies against Meaban virus (0 %; 95 %CI: 0.0-0.7 %) were not found in the tested sera. The results demonstrate WNV, USUV and TBEV exposure in zoo mammals, which may be of public health and conservation concern. Seropositivity to WNV and USUV was detected in regions where these viruses have not been reported previously. Anti-WNV antibodies found in zoo animals sampled in 2009 point to WNV circulation at least one year before the first outbreaks were reported in horses and humans in Spain. Our results indicate that zoo mammals could be useful sentinel species for monitoring emerging flavivirus activity in urban areas.

Usutu virus is an emerging mosquito-borne flavivirus initially identified in South Africa in 1959 that is now circulating throughout parts of Africa, Europe, and the Middle East. It is closely related to West Nile virus, and has similar vectors, amplifying bird hosts, and epidemiology. Usutu virus infection can occur in humans and may be asymptomatic or cause systemic (e.g., fever, rash, and hepatitis) or neuroinvasive (e.g., meningitis and encephalitis) disease. Given few reported cases, the full clinical spectrum is not known. No anti-viral treatment is available, but it can be largely prevented by avoiding mosquito bites. Because of similar mosquitoes, birds, and climate to Europe, the potential for introduction to North America is possible.

The intensification of trade and travel is linked to the growing number of imported cases of dengue, chikungunya or Zika viruses into continental Europe and to the expansion of invasive mosquito species such as Aedes albopictus and Aedes japonicus. Local outbreaks have already occurred in several European countries. Very little information exists on the vector competence of native mosquitoes for arboviruses. As such, the vectorial status of the nine mosquito species largely established in North-Western Europe (Aedes cinereus and Aedes geminus, Aedes cantans, Aedes punctor, Aedes rusticus, Anopheles claviger s.s., Anopheles plumbeus, Coquillettidia richiardii, Culex pipiens s.l., and Culiseta annulata) remains mostly unknown.
To review the vector competence of both invasive and native mosquito populations found in North-Western Europe (i.e., France, Belgium, Germany, United Kingdom, Ireland, The Netherlands, Luxembourg and Switzerland) for dengue, chikungunya, Zika, West Nile and Usutu viruses.
A bibliographical search with research strings addressing mosquito vector competence for considered countries was performed.
Out of 6357 results, 119 references were related to the vector competence of mosquitoes in Western Europe. Eight species appear to be competent for at least one virus.
Aedes albopictus is responsible for the current outbreaks. The spread of Aedes albopictus and Aedes japonicus increases the risk of the autochthonous transmission of these viruses. Although native species could contribute to their transmission, more studies are still needed to assess that risk.