West Nile or Usutu Virus? A Three-Year Follow-Up of Humoral and Cellular Response in a Group of Asymptomatic Blood Donors

Abstract

West Nile virus (WNV) and Usutu virus (USUV) are two related arboviruses (genus Flavivirus, family Flaviviridae), with birds as a reservoir and mosquitoes as transmitting vectors. In recent years, WNV epidemiology changed in many European countries with increased frequency of outbreaks posing the issue of virus transmission risks by blood transfusion. USUV emerged for the first time in birds of the Tuscany region (Italy) in 1996 and in 2001 in Austria. While WNV is responsible for both mild and neuroinvasive diseases, USUV infection is usually asymptomatic and neuroinvasive symptoms are rare. Since WNV and USUV co-circulate, the surveillance of WNV allows also the detection of USUV. Due to the great similarity in amino-acid sequence of major surface proteins of the two viruses, a high cross-reactivity can lead to misinterpretation of serological results. Here, we report the results obtained from 54 asymptomatic blood donors during a three-year follow-up showing an unexpected high positivity (46.3%) for USUV. The major obstacle encountered in the differential diagnosis between these two viruses was the high cross-reactivity found in neutralizing antibodies (NT Abs) and, in some cases, a long follow-up was mandatory for a correct diagnosis. Moreover, two new ELISpot assays were developed for a more rapid and specific differential diagnosis, especially in those cases in which NT Abs were not determinant. Using a combination of Enzyme-linked immunospot (ELISpot), molecular, and serological tests, we could identify 25 true positive WNV and 25 true positive USUV blood donors. Our data highlight the importance of raising awareness for increasing USUV infections in endemic countries involved in blood transfusion and organ donation.

Prevalence of SARS-CoV-2 specific neutralising antibodies in blood donors from the Lodi Red Zone in Lombardy, Italy, as at 06 April 2020

“…An in-house microneutralisation assay adapted to SARS-CoV-2 from a previously reported method was employed [11]. In addition, specific real-time RT-PCRs targeting RNA-dependent RNA polymerase and envelope (E) genes were used to detect the presence of SARS-CoV-2 according to the World Health Organization guidelines [12] and the Corman et al protocol [13].…”

Epidemiology of Usutu Virus: The European Scenario

“…In 2014–2015, a very high USUV seroprevalence was found in forestry workers (18.1%) compared to blood donors (1%) in the area of the Po river valley (northern Italy) [ 96 ]. During 2016–2018, an unexpected high seropositivity (46.3%) for USUV was found in asymptomatic blood donors in the Lombardy region [ 97 ]. In 2017 and 2018, five blood donations tested in the Lazio region were USUV RNA positive with a circulation of different USUV strains (Europe 2, 3 and 4 lineages) [ 69 ].…”

Development and characterization of specific anti‐Usutu virus chicken‐derived single chain variable fragment antibodies

“…[20][21][22] Indeed, WNV and USUV have been misdiagnosed for each other 23 and several infections were falsely attributed to WNV before thorough genetic reevaluation. 24,25 The high cross-reactivity found in antibodies is still a challenge for differential diagnosis of infections by USUV and WNV. 26,27 Further, such crossreactivity has been linked to severe sickness via antibodydependent enhancement in sequential infections of different flaviviruses.…”

“…26,27 Further, such crossreactivity has been linked to severe sickness via antibodydependent enhancement in sequential infections of different flaviviruses. 25 The cross-reactivity among flaviviruses, especially between the co-circulating WNV and USUV, and the current lack of reliable commercial tests for USUV may have resulted into a vastly underestimated presence of USUV in Europe. Its low prevalence in the human population bears the risk of a potential outbreak in Europe.…”

“…These circumstances, along with extensive cross‐reactivity among flaviviruses owing to sequence similarity in the envelope (E) protein, might have led to misinterpretation of serological test results 20–22 . Indeed, WNV and USUV have been misdiagnosed for each other 23 and several infections were falsely attributed to WNV before thorough genetic re‐evaluation 24,25 . The high cross‐reactivity found in antibodies is still a challenge for differential diagnosis of infections by USUV and WNV 26,27 .…”