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A new strain of WNV lineage 1 (WNV - 1) emerged in the Veneto Region, northern Italy, in 2021, eight years after the last outbreak of WNV - 1 in Italy. The virus, which co-circulates with WNV-2, has become endemic in the Region, where, in 2022, most human cases of neuroinvasive disease (WNND) reported in Europe have occurred.Comparative analysis of the epidemiology and clinical presentation of WNV-1 and WNV-2 infection in humans, as well as the temporal and geographic distribution of WNV-1 and WNV-2 among wild birds and Culex pipiens mosquitoes in Veneto, from May 16th to August 21st, 2022, to determine if the high number of WNND cases was associated with WNV-1.As of August 21st, 2022, 222 human cases of WNV infection were confirmed by molecular testing, including 103 with fever (WNF) and 119 with WNND. WNV lineage was determined in 201 (90.5%) cases, comprising 138 WNV-1 and 63 WNV-2 infections. During the same period, 35 blood donors tested positive, including 30 in whom WNV lineage was determined (13 WNV-1 and 17 WNV-2). Comparative analysis of the distribution of WNV-1 and WNV-2 infections among WNND cases, WNF cases and WNV-positive blood donors showed that patients with WNND were more likely to have WNV-1 infection than blood donors (odds ratio 3.44; 95% CI 95% 1.54 to 8.24; p = 0.0043). As observed in humans, in wild birds WNV-1 had higher infectious rate (IR) and showed a more rapid expansion than WNV-2. At variance, the distribution of the two lineages was more even in mosquitoes, but with a trend of rapid increase of WNV-1 IR over WNV-2.Comparative analysis of WNV-1 vs WNV-2 infection in humans, wild birds, and mosquitos showed a rapid expansion of WNV-1 and suggested that WNV-1 infected patients might have an increased risk to develop severe disease.

Additional file 2: Figure S1. DHARMa residual diagnostics plots [39] (qq-plot and residuals plotted against the predicted value) for the Poisson regression model (upper plots) and the negative binomial regression model (lower plots) for Anopheles messeae/Anopheles daciae. Tests for correct distribution (KS test), dispersion and outliers are shown on the plots.

Additional file 3: Table S3. Shapiro–Wilk test and Levene’s test to check requirements of normality and homogeneity of variance across groups, respectively.

Additional file 4: Table S4. Incident rate ratios (i.e. exponential of coefficient estimates), 95% CIs and significance for Culex pipiens and Ochlerotatus caspius. Table S5. Contrasts between trapping devices for estimated marginal means after model fitting, considering a confidence level of 0.95, for Culex pipiens and Ochlerotatus caspius. Estimates are back-transformed from the log scale.

Additional file 1: Table S1. Sampling sites and climatic data (temperature and precipitation) for the sampling period (trapping cycle). Table S2. Numbers and percentages of adults of species of the Anopheles maculipennis complex molecularly identified, according to collection method.

Additional file 1: Table S1. Classification of the mosquito trapping sites per year, based on the land cover classes (level 2 of the Corine Land Cover 2018) where the traps were located. Figure S1.Post-hoc Tukey’s comparisons between years for average mosquito abundance, density-dependent variable and environmental variables. Figure S2. Annual trends of the level of aridity (DMI), normalized difference vegetation index (NDVI) and temperature (maximum, red line; minimum, blue line, LST) from 2010 to 2018. Gray lines: monthly averages for the whole study period. Table S2. Correlation matrix of intrinsic and extrinsic variables included in the analyses (absolute correlation values > 0.65 are reported in boldface).

Additional file 2: Table S3. Lists of models per year included in the subsets considered in the study; models are ordered by decreasing wAICc and likelihood-ratio-based pseudo-R2 ($$ R^{2}{_{\text{LR}} }$$R2LR) is reported to indicate the goodness-of-fit of each model.