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Virus research2025; 355; 199557; doi: 10.1016/j.virusres.2025.199557

Is it time to consider west Nile and Usutu viruses endemic in central Italy?

Abstract: West Nile (WNV) and Usutu (USUV) viruses co-circulated in a region of Central Italy (Lazio) in 2018, as evidenced by the detection of WNV in the nervous tissues of symptomatic horses and USUV in blood donors and mosquito pools. To assess whether these viruses were endemic in the region, we analysed: 1) diapausing Culex pipiens mosquitoes collected during the winter seasons 2022-2023 and 2023-2024, 2) Cx. pipiens mosquitoes collected during the adult activity period from April to November in 2022 and 2023 across 4 provinces, and 3) sera from 52 horses and tissues from 537 birds. Field-collected Cx. pipiens, including both diapausing and non-diapausing individuals, were tested in pools for WNV and USUV using real-time RT-PCR. Serum samples from horses were tested with two WNV ELISA assays, IgM and IgG, while bird tissues were tested for both viruses via real-time RT-PCR. A total of 18,834 Cx. pipiens females were collected, including 9,812 mosquitoes during the winter seasons and 9,022 during the adult activity periods. Mosquitoes were tested in 623 pools, with all pools of diapausing mosquitoes testing negative for both viruses and 12 pools of non-diapausing mosquitoes positive to USUV. The WNV IgG positivity of 7 horse sera, which were negative at the beginning of the study period, was not confirmed by the virus neutralization test. All tissue samples were negative for WNV and USUV. Since WNV and USUV were not detected in diapausing mosquitoes, there was no evidence of the two viruses endemicity in the study area.
Copyright © 2025. Published by Elsevier B.V.
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Publication Date: 2025-03-11 PubMed ID: 40081763PubMed Central: PMC11957532DOI: 10.1016/j.virusres.2025.199557Google Scholar: Lookup
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

Overview

  • This research investigated whether West Nile virus (WNV) and Usutu virus (USUV) have become endemic in central Italy by testing mosquitoes, horses, and birds for evidence of the viruses over multiple years and seasons.
  • Although USUV was detected in active mosquitoes, the viruses were not found in diapausing (overwintering) mosquitoes or in animal samples, suggesting these viruses may not be permanently established in the region.

Background and Objective

  • West Nile virus (WNV) and Usutu virus (USUV) are arboviruses transmitted primarily by Culex mosquitoes, affecting birds, horses, and humans.
  • Both viruses were detected co-circulating in the Lazio region of central Italy in 2018, raising questions about their potential endemicity—that is, whether they persist locally year-round.
  • The study’s main goal was to determine if WNV and USUV were endemic in central Italy by analyzing mosquito populations throughout different seasons and testing potential reservoirs such as horses and birds.

Study Design and Methods

  • Sampling focused on different seasons and host/vector groups:
    • Diapausing (overwintering) Culex pipiens mosquitoes collected during winters of 2022-2023 and 2023-2024.
    • Non-diapausing (active adult) Culex pipiens mosquitoes collected April to November 2022 and 2023 in four provinces.
    • Serum samples from 52 horses to assess virus exposure.
    • Tissue samples from 537 birds to identify active infections.
  • Mosquitoes were grouped into pools and tested via real-time reverse transcription PCR (RT-PCR) to detect WNV and USUV RNA.
  • Horse sera were tested using two ELISA assays targeting WNV-specific IgM and IgG antibodies, with confirmation attempted via virus neutralization tests to verify true exposure.
  • Bird tissue samples underwent RT-PCR testing for both viruses.

Key Findings

  • A total of 18,834 female Cx. pipiens mosquitoes were collected:
    • 9,812 during the winter/senescing periods (diapausing).
    • 9,022 during the adult activity periods (spring to fall).
  • In 623 mosquito pools tested:
    • All pools of diapausing mosquitoes were negative for both WNV and USUV, indicating the viruses are likely not overwintering inside local mosquitoes.
    • Twelve pools of non-diapausing mosquitoes were positive for USUV, showing active circulation during warmer months.
  • Among horse sera, seven samples showed WNV IgG positivity initially but these results were not confirmed by the more specific virus neutralization tests, suggesting possible false positives or non-specific antibodies.
  • All 537 bird tissue samples tested negative for WNV and USUV, showing no evidence of active infections in avian hosts at the time of sampling.

Interpretation and Implications

  • The absence of virus RNA in diapausing mosquitoes suggests the viruses do not persist locally during winter months within mosquito populations, which is a key factor for establishing endemicity.
  • The detection of USUV in active mosquitoes during warmer months shows seasonal virus circulation but not necessarily year-round maintenance.
  • Negative results in birds and lack of confirmed seropositivity in horses further support the conclusion that the viruses have not established permanent cycles in this region.
  • This study implies that WNV and USUV are likely introduced seasonally rather than maintained year-round in central Italy, and the area may be experiencing episodic outbreaks rather than endemic transmission.
  • Continued surveillance is needed due to the public health importance of these viruses and changing ecological conditions that could favor future endemicity.

Conclusions

  • No evidence was found to support the status of WNV and USUV as endemic viruses in central Italy based on mosquito overwintering, horse serology, and bird testing.
  • The viruses appear to circulate seasonally during the mosquito active periods but do not persist through the winter cycle in this region.
  • These findings help shape local public health strategies, emphasizing seasonal monitoring and vector control rather than sustained measures against endemic disease.

Cite This Article

APA
Romiti F, Scicluna MT, Censi F, Micarelli F, Puccica S, Carvelli A, Sala MG, Del Lesto I, Casini R, De Liberato C, Tofani S. (2025). Is it time to consider west Nile and Usutu viruses endemic in central Italy? Virus Res, 355, 199557. https://doi.org/10.1016/j.virusres.2025.199557

Publication

Virus research
ISSN: 1872-7492
NlmUniqueID: 8410979
Country: Netherlands
Language: English
Volume: 355
Pages: 199557
PII: 199557

Researcher Affiliations

Romiti, Federico
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy. Electronic address: federico.romiti@izslt.it.
Scicluna, Maria Teresa
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Censi, Francesco
  • ASL Latina, Sanità Animale e Igiene degli Allevamenti, Via Nettunense, 04011 Aprilia (LT), Italy.
Micarelli, Florindo
  • ASL Latina, Sanità Animale e Igiene degli Allevamenti, Via Nettunense, 04011 Aprilia (LT), Italy.
Puccica, Silvia
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Carvelli, Andrea
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Sala, Marcello Giovanni
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Del Lesto, Irene
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Casini, Riccardo
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
De Liberato, Claudio
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.
Tofani, Silvia
  • Istituto Zooprofilattico Sperimentale del Lazio e della Toscana M. Aleandri, Via Appia Nuova 1411, 00178, Rome, Italy.

MeSH Terms

  • Animals
  • Italy / epidemiology
  • Horses / virology
  • West Nile virus / isolation & purification
  • West Nile virus / genetics
  • West Nile virus / immunology
  • Flavivirus / isolation & purification
  • Flavivirus / genetics
  • Flavivirus / immunology
  • Culex / virology
  • West Nile Fever / epidemiology
  • West Nile Fever / veterinary
  • West Nile Fever / virology
  • Flavivirus Infections / epidemiology
  • Flavivirus Infections / veterinary
  • Flavivirus Infections / virology
  • Horse Diseases / epidemiology
  • Horse Diseases / virology
  • Birds / virology
  • Antibodies, Viral / blood
  • Female
  • Mosquito Vectors / virology
  • Endemic Diseases / veterinary
  • Seasons
  • Immunoglobulin G / blood

Conflict of Interest Statement

Declaration of competing interest The authors declare the following financial interests/personal relationships: Federico Romiti reports financial support, equipment, drugs, or supplies, and travel were provided by Italian Ministry of Health. Silvia Tofani reports equipment, drugs, or supplies and travel were provided by Italian Ministry of Health. Marcello Giovanni Sala reports article publishing charges and travel were provided by Italian Ministry of Health. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Citations

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