Lack of identification of Flaviviruses in oral and cloacal swabs from long- and short-distance migratory birds in Trentino-Alto Adige (North-eastern Italy)

Abstract

BackgroundWest Nile virus (WNV) and Usutu virus (USUV), both belonging to the genus Flavivirus, are emerging in Italy as important human and animal pathogens. Migratory birds are involved in the spread of Flaviviruses over long distances, particularly from Africa to Europe. Once introduced, these viruses can be further be dispersed by short-distance migratory and resident bird species. Thus far, there is still a considerable knowledge gap on the role played by different bird species in the ecology and transmission mechanisms of these viruses. The Region of Trentino-Alto Adige (north-eastern Italy) is located on the migratory route of many of the short- and long-distance migratory birds that cross the Alps, connecting northern Europe and western Asia with southern Europe and Africa. Until now, only a silent circulation of WNV and USUV within the territory of the Province of Trento has been confirmed by serological screening, whilst no cases of infected humans or animals have so far been reported. However, continuous spillover events of both viruses have been reported in neighbouring Regions. The aim of this study was to monitor the circulation of WNV and USUV in Trentino-Alto Adige, in order to detect if active virus shedding occurs in migratory birds captured during their seasonal movements and to evaluate the role that different bird species could play in the spreading of these viruses.MethodsWe carried out a biomolecular survey on oral and cloacal swabs collected from migratory birds during seasonal migrations. Birds belonging to 18 transaharian and 21 intrapaleartic species were examined during spring (n = 176) and autumn (n = 146), and were tested using a generic nested-PCR.ResultsAll samples tested negative for Flaviviruses. The possible causes of unapparent shedding, along with ecological and epidemiological implications are discussed.ConclusionsThe lack of detection of active virus shedding in these bird species does not exclude the circulation of these viruses within the Trentino-Alto Adige region, as reported in previous studies. The possible ecological implications are discussed.

Lack of usutu virus RNA in cerebrospinal fluid of patients with encephalitis of unknown etiology, Tuscany, Italy

“…As a consequence, an intense WNV surveillance was implemented in that area involving Emilia‐Romagna, Veneto and Lombardy. Simultaneous circulation of USUV was detected by testing wild birds found dead and mosquitoes [Calzolari et al, ; Busani et al, ; Tamba et al, ] and sentinel horses and chickens [Savini et al, ] in North Eastern Italy, excluding Trentino–Alto Adige [Grisenti et al, ]. Recently, retrospective analysis of archived tissue samples from Eurasian blackbirds ( Turdus merula ) deaths in the Tuscany region (Florence and Pistoia provinces) of Italy in 1996 identified USUV.…”

Flaviviruses in migratory passerines during spring stopover in a desert oasis

“…We also carried out a generic pan‐flavivirus RT‐PCR for screening of flavivirus presence. We used QIAGEN one‐step RT‐PCR kit and primers that amplify a region of the NS5 gene which is well conserved within this genus (Grisenti et al, ). Primers used were WNV9040 (5′TACAACATGATGGGVAARAGAGAGA‐3′) and WNV10124 (5‐AGCATGTCTTCYGTBGTCATCCAYT‐3), resulting in a 1,084‐bp amplification product (Weissenbock et al, ).…”

“…In particular, oral swab samples generally contain more virus particles than cloacal ones (Guerrero‐Sanchez et al, ). Thereby, birds identified as positive could be suspected to be involved in infecting local mosquitoes or passively shedding the viruses in nature, but this may depend on the viremia (Grisenti et al, ; Komar et al, ; Wheeler, Vineyard, Barker, & Reisen, ). Serological tools allow assessment of whether a bird has been in contact with the viruses during its lifetime through the detection of specific antibodies (Nemeth et al, ).…”

“…Positive USUV serology has been reported in some migratory passerines, such as Sylvia communis , Sylvia borin , Ficedula hypoleuca and Hirundo rustica (Ashraf et al, ; Meister et al, ). However, few investigations have been carried out to detect USUV oro‐faecal shedding in migratory passerines, and the results have always been negative (Grisenti et al, ).…”

Wide detection of Aedes flavivirus in north-eastern Italy – a European hotspot of emerging mosquito-borne diseases

“…Moreover, DNA sequences related to AeFV were identified in wild Aedes albopictus mosquito collected in 2007(Roiz et al, 2009). In Trentino, evidence of WNV and USUV circulation has been detected sporadically through seroconversion of sentinel chickens in 2005, but no evidence of current active virus shedding from birds or occurrence in mosquitoes has been recorded so far (Rizzoli et al, 2007;Grisenti et al, 2013). A completely different eco-epidemiological situation characterizes Veneto, where WNV (Filipponi et al, 2005(Filipponi et al, , 2007Calistri et al, 2010; Barzon et al, 2013a, b) and USUV (Regione del Veneto, 2011 Busani et al, 2011;Savini et al, 2011;Engler et al, 2013;Gobbi et al, 2014) have been detected on several occasions during the past 10 years, and now circulate endemically with periodical outbreaks.…”